Transmitting/Reflecting Emanating Light With Time Variation

ABSTRACT

A filter arrangement can transmit and/or reflect light emanating from a moving object so that the emanating light has time variation, and the time variation can include information about the object, such as its type. For example, emanating light from segments of a path can be transmitted/reflected through positions of a filter assembly, and the transmission functions of the positions can be sufficiently different that time variation occurs in the emanating light between segments. Or emanating light from a segment can be transmitted/reflected through a filter component in which simpler transmission functions are superimposed, so that time variation occurs in the emanating light in accordance with superposition of two simpler non-uniform transmission functions. Many filter arrangements could be used, e.g. the filter component could include the filter assembly, which can have one of the simpler non-uniform transmission functions. Time-varying waveforms from sensing results can be compared to obtain spectral differences. The filter arrangement, in a practical commercial embodiment, can be manufactured to be disposable, and used in a point-of-care device for use practically anywhere, at low cost, and can also be implemented in an in-line monitoring system.

CLAIM FOR PRIORITY

Pursuant to 37 C.F.R. §§1.52(b)(2) and 1.78, this application claimspriority as a Continuation-in-Part Application under 35 U.S.C. §120 toU.S. Non-provisional Patent Application entitled“TRANSMITTING/REFLECTING EMANATING LIGHT WITH TIME VARIATION”, filed inthe U.S. Patent and Trademark Office on Feb. 1, 2008, and assigned Ser.No. 12/024,490, the entire contents of which are expressly incorporatedherein by reference.

CROSS REFERENCE TO RELATED APPLICATIONS

The following applications, each of which is hereby incorporated byreference in its entirety, might be regarded as related to thisapplication: “Additive Printed Mask Process and Structures ProducedThereby”, U.S. patent application Ser. No. 10/536,102, now published asU.S. Patent Publication No. 2007/0172969; “Sensing Photon EnergiesEmanating from Channels or Moving Objects”, U.S. patent application Ser.No. 11/315,386, now published as U.S. Patent Publication No.2007/0146704; “Sensing Photon Energies of Optical Signals”, U.S. patentapplication Ser. No. 11/315,926, now published as U.S. PatentPublication No. 2007/0147189; “Sensing Photons from Objects inChannels”, U.S. patent application Ser. No. 11/315,992, now published asU.S. Patent Publication No. 2007/0145249; “Obtaining AnalyteInformation”, U.S. patent application Ser. No. 11/316,303, now publishedas U.S. Patent Publication No. 2007/0148760; “Providing Light toChannels or Portions”, U.S. patent application Ser. No. 11/316,660, nowpublished as U.S. Patent Publication No. 2007/0147728; “Method andSystem for Evaluation of Signals Received from Spatially ModulatedExcitation and Emission to Accurately Determine Particle Positions andDistances”, U.S. patent application Ser. No. 11/698,338; “Method andSystem Implementing Spatially Modulated Excitation or Emission forParticle Characterization with Enhanced Sensitivity”, U.S. patentapplication Ser. No. 11/698,409; “Obtaining Information From OpticalCavity Output Light”, U.S. patent application Ser. No. 11/702,249;“Photosensing Optical Cavity Output Light”, U.S. patent application Ser.No. 11/702,250; “Distinguishing Objects”, U.S. patent application Ser.No. 11/702,328; “Encoding Optical Cavity Output Light”, U.S. patentapplication Ser. No. 11/702,363; “Moving Analytes and Photosensors”,U.S. patent application Ser. No. 11/702,470; “Surface Energy ControlMethods for Color Filter Printing”, U.S. patent application Ser. No.11/755,717; “Producing Sandwich Waveguides”, U.S. patent applicationSer. No. 11/777,661; “Producing Fluidic Waveguides”, U.S. patentapplication Ser. No. 11/777,712; “Obtaining Information from TimeVariation of Sensing Results”, U.S. patent application Ser. No.12/022,485; and “Providing Time Variation in Emanating Light”, U.S.patent application Ser. No. 12/023,436.

BACKGROUND OF THE INVENTION

The present invention relates generally to techniques that transmitand/or reflect light emanating from objects. More specifically,techniques can use filter arrangements to transmit and/or reflect suchlight with time variation, such as where the objects are moving relativeto the filter arrangements.

Various techniques have been proposed for using light emanating fromobjects. For example, U.S. Patent Application Publication No.2007/0145249 describes a fluidic structure with a channel along which isa series of sensing components to obtain information about objectstraveling within the channel, such as droplets or other objects carriedby fluid. A sensing component includes a set of cells that photosense arange of photon energies that emanate from objects. A processor canreceive information about objects from the sensing components and use itto obtain spectral information. Similar techniques are described, forexample, in U.S. Patent Application Publication Nos. 2007/016704,2007/0147189, and 2007/0147728.

Also, various flow cytometry techniques have been proposed.

It would be advantageous to have improved techniques for using lightemanating from objects, including improved techniques for transmittingand/or reflecting such light with time variation.

SUMMARY OF THE INVENTION

The invention provides various exemplary embodiments, including methodsand apparatus. In general, the embodiments involve transmitting and/orreflecting emanating light through filter arrangements to obtain timevariation.

All the above described disadvantages are overcome and a number ofadvantages are realized by a first aspect that relates to an article ofmanufacture comprising: a fluid-engaging structure, wherein thefluid-engaging structure includes (a) a channel that in use can containfluid and through which objects can move; (b) one or more bounding partsthat bound the channel, and wherein at least one of the one or morebounding parts include one or more light transmissive portions, whereinat least one of the one or more light transmissive portions isconfigured to receive excitation light and provide the receivedexcitation light, and wherein excitation light enters the channel andinteracts with an object resulting in emanating light; and (c) one ormore mask arrangements configured to receive at least part of theemanating light and in response, provide encoded emanating light,wherein the one or more mask arrangements and the channel are furtherconfigured so that the encoded emanating light includes time variationresulting from relative movement between the one or more maskarrangements and the object, the time variation including informationabout the object.

According to the first aspect, the a method of using the article ofmanufacture is provided comprising causing an object to move through thechannel in a fluid; causing the excitation light to enter the channelthrough the one or more light transmissive portions and interact withthe object, resulting in the emanating light from the object; receivingthe emanating light at a first of the one or more mask arrangements; andproviding the encoded emanating light with the time variation includingthe information about the object in response to the received emanatinglight.

Still further according to the first aspect, the fluid is water and theobject includes at least one of e. coli, giardia, cryptosporidium, andbacillus endospore, and the fluid is human blood and the object includesat least one of CD4 lymphocytes and CD4 monocytes.

According to the first aspect, the method further comprises receivingthe encoded emanating light at a photosensitive surface of a large areaphotosensor; and providing an electrical signal by the large areaphotosensor, in response to the received encoded emanating light, theelectrical signal indicating one or more sensed time-varying waveforms,and wherein at least one of the one or more sensed time-varyingwaveforms indicating the information about the object. According to thefirst aspect, a host structure includes the large area photosensor, thehost structure being separate from and useable with the fluid-engagingstructure, the method further comprising operating circuitry in the hoststructure to respond to the electrical signal by providing dataindicating the information about the object.

According to the first aspect, the host structure includes one of ahandheld device, the handheld device configured to monitor discretesamples of fluid and objects, and an in-line device, the in-line deviceconfigured to substantially continuously monitor the fluid and objects,and wherein the fluid-engaging structure further comprises: a first ofthe one or more mask arrangements configured to receive at least part ofthe emanating light and in response provide encoded emanating light toreceived emanating light, and is located on a first portion of thebounding parts; one or more mirrors, at least one of the one or moreminors configured to substantially reflect the excitation light throughthe one or more light transmissive portions; and an internal reflectingsurface, wherein the internal reflecting surface is configured tore-transmit the reflected excitation light into the channel with thefluid and objects, and wherein the article of manufacturing isconfigured for use with a host structure, and wherein the host structureincludes: at least one excitation light source; a filter configured toreceive and filter the encoded emanating light; and a photo-sensorconfigured to receive the filtered encoded emanating light and detecttime variation resulting from relative movement between the one or moremask arrangements and the objects, the time variation includinginformation about the objects, and wherein the photo-sensor is furtherconfigured to provide an electrical signal indicating one or more sensedtime-varying waveforms.

Still further according to the first aspect, the article of manufactureis configured to be disposable, the excitation light source includes oneof a light emitting diode and laser diode, and the photo sensor is oneof a large area photo sensor and a PIN diode. According to the firstaspect, the article of manufacturing is configured for use with a hoststructure, and wherein the host structure comprises: at least oneexcitation light source; at least one filter configured to receive andfilter the encoded emanating light; and a photo-sensor configured toreceive the filtered encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the photo-sensor and the at least onefilter configured to receive and filter the encoded emanating light arelocated on a first exterior side of the one or more bounding parts ofthe fluid-engaging structure, and the at least one excitation lightsource is located substantially directly opposite that of thephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light.

According to the first aspect, the article of manufacturing isconfigured for use with a host structure, and wherein the host structurecomprises: at least one excitation light source; at least one filterconfigured to receive and filter the encoded emanating light; and aphoto-sensor configured to receive the filtered encoded emanating lightand detect time variation resulting from relative movement between theone or more mask arrangements and the objects, the time variationincluding information about the objects, and wherein the photo-sensorand the at least one filter configured to receive and filter the encodedemanating light are located on a first exterior side of the one or morebounding parts of the fluid-engaging structure, and the at least oneexcitation light source is located substantially adjacent to that of thephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light.

Still further according to the first aspect, the fluid-engagingstructure further comprises: at least two mirrors, wherein a firstmirror is configured to substantially reflect first excitation lightfrom a first excitation light source through the one or more lighttransmissive portions, and a second mirror is configured tosubstantially reflect second excitation light from a second excitationlight source through the one or more light transmissive portions; and aninternal reflecting surface, wherein the internal reflecting surface isconfigured to re-transmit the first reflected excitation light into thechannel with the fluid and objects, and the internal reflecting surfaceis configured to re-transmit the second reflected excitation light intothe channel with the fluid and objects, and wherein the re-transmittedfirst and second excitation light interacts with the object resulting ina combined emanating light; and a first of the one or more maskarrangements is located on a first portion of the bounding parts,wherein, the first of the one or more mask arrangements is configured toreceive at least part of the combined emanating light at a first rangeof photon energies and, in response, provide encoded combined emanatinglight at the first range of photon energies.

According to the first aspect, the article of manufacturing furthercomprises a host structure, the host structure including the firstexcitation light source configured to transmit the first excitationlight; the second excitation light source configured to transmit thesecond excitation light; a first photo-sensor; a second photo-sensor; asecond of the one or more mask arrangements, and wherein the second ofthe one or more mask arrangements is configured to receive at least partof the combined emanating light and, in response, provide encodedcombined emanating light at a second range of photon energies; a firstfilter located substantially adjacent to the first photo-sensor, whereinthe first filter is configured to pass a first portion of the encodedcombined emanating light that corresponds to the first range of photonenergies to the first photo-sensor; and a second filter locatedsubstantially adjacent to the second photo-sensor, wherein the secondfilter is configured to pass a second portion of the encoded combinedemanating light that corresponds to the second range of photon energiesto the second photo-sensor.

According to the first aspect, the first photo-sensor is configured toreceive the filtered first portion of the encoded combined emanatinglight that corresponds to the first range of photon energies and detecttime variation resulting from relative movement between the one or moremask arrangements and the objects, the time variation includinginformation about the objects, and wherein the first photo-sensor isfurther configured to provide a first set of electrical signalsindicating one or more sensed time-varying waveforms to host structurecircuitry, and the second photo-sensor is configured to receive thefiltered second portion of the encoded combined emanating light thatcorresponds to the second range of photon energies and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a second set of electrical signals indicating oneor more sensed time-varying waveforms to host circuitry.

According to the first aspect, the first and second excitation lightsources includes one of a laser diode and light emitting diode, and thehost structure further comprises: an optical device for re-directingencoded combined emanating light at either a first range of photonenergies or at a second range of photon energies.

According to the first aspect, the fluid-engaging structure furthercomprises: a first receptacle for accepting the fluid, and the objectswithin the fluid; a second receptacle for accepting the fluid and theobjects within the fluid following passage through the channel; and avacuum spring-loaded syringe configured to compel movement of the fluidand the objects through the channel, and to energize an excitation lightsource upon release of the spring.

According to the first aspect, the fluid-engaging structure comprises: afirst of the one or more mask arrangements is configured to receive atleast part of the emanating light and, in response, providecolor-dependent encoded emanating light and is located on a firstportion of the bounding parts, and wherein the fluid engaging structureis configured for use with a host structure, and wherein the hoststructure includes: at least one excitation light source; a filterconfigured to receive and filter the color-dependent encoded emanatinglight; and a photo-sensor configured to receive the filteredcolor-dependent encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the photo-sensor is further configured toprovide an electrical signal indicating one or more sensed time-varyingwaveforms.

Still further according to the first aspect, the fluid-engagingstructure comprises: a first of the one or more mask arrangementsconfigured to receive at least part of the emanating light and, inresponse, provide first color-dependent encoded emanating light and islocated on a first portion of the bounding parts; a second of the one ormore mask arrangements configured to receive at least part of theemanating light and, in response, provide second color-dependent encodedemanating light and is located on a second portion of the boundingparts, and wherein the fluid engaging structure is configured for usewith a host structure, and wherein the host structure includes: a firstexcitation light source; a second excitation light source; a firstfilter configured to receive and filter the first color-dependentencoded emanating light, the first filter located substantially adjacentto the first of the one or more mask arrangements; a second filterconfigured to receive and filter the second color-dependent encodedemanating light, the second filter located substantially adjacent to thesecond of the one or more mask arrangements; a first photo-sensorlocated substantially adjacent to the first filter, and wherein thefirst photo-sensor is configured to receive the first filteredcolor-dependent encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the first photo-sensor is furtherconfigured to provide a first electrical signal indicating one or moresensed time-varying waveforms; and a second photo-sensor locatedsubstantially adjacent to the second filter, and wherein the secondphoto-sensor is configured to receive the second filteredcolor-dependent encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a second electrical signal indicating one or moresensed time-varying waveforms.

A second aspect that overcomes all of the above described disadvantagesand provides a number of advantages includes an article of manufacturecomprising: a host structure, wherein the host structure includes (a) atleast one excitation light source; (b) one or more support partsstructured to support a fluid-engaging structure on the host structure,wherein the fluid-engaging structure includes a channel that in use cancontain fluid and through which objects can move; one or more boundingparts that bound the channel, and wherein at least one of the one ormore bounding parts include one or more light transmissive portions,wherein at least one of the one or more light transmissive portions isconfigured to receive excitation light and provide the receivedexcitation light, and wherein excitation light enters the channel andinteracts with an object resulting in emanating light; and one or moremask arrangements configured to receive at least part of the emanatinglight and, in response, provide encoded emanating light, and wherein theone or more mask arrangements and the channel are further configured sothat the encoded emanating light includes time variation resulting fromrelative movement between the one or more mask arrangements and theobject, the time variation including information about the object; (c)at least one filter configured to receive and filter the encodedemanating light; (d) at least one photo-sensor configured to receive thefiltered encoded emanating light and detect time variation resultingfrom relative movement between the one or more mask arrangements and theobjects, the time variation including information about the objects, andwherein the photo-sensor is further configured to provide a firstelectrical signal indicating one or more sensed time-varying waveforms;and (e) circuitry configured to receive the first electrical signals andwhich is further configured to provide second electrical signals inresponse to the first electrical signals resulting from photosensing ofthe encoded emanating light, and wherein the second electrical signalsindicate the information about the objects.

According to the second aspect, the host structure is configured for useas a point-of-care apparatus, the excitation light source includes oneof a light emitting diode and laser diode, the at least one photo-sensoris one of a large area photo sensor and a PIN diode, and thephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light are located on a first exterior sideof the one or more bounding parts of the fluid-engaging structure, andthe at least one excitation light source is located substantiallydirectly opposite that of the photo-sensor and the at least one filterconfigured to receive and filter the encoded emanating light.

Still further according to the second aspect, the at least onephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light are located on a first exterior sideof the one or more bounding parts of the fluid-engaging structure, andthe at least one excitation light source is located substantiallyadjacent to that of the photo-sensor and the at least one filterconfigured to receive and filter the encoded emanating light, and thehost structure further comprises: a first excitation light sourceconfigured to transmit first excitation light; a second excitation lightsource configured to transmit the second excitation light, and whereinthe fluid-engaging structure is configured to provide combined emanatinglight resulting from interaction between the first excitation light andthe objects, and the second excitation light and the objects, andwherein the fluid-engaging structure further includes a first of the oneor more mask arrangements that is located on a first portion of thebounding parts, and wherein, the first of the one or more maskarrangements is configured to receive at least part of the combinedemanating light at a first range of photon energies and, in response,provide encoded combined emanating light, the host structure furthercomprising; a first photo-sensor; a second photo-sensor; a second of theone or more mask arrangements, and wherein the second of the one or moremask arrangements is configured to receive at least part of the combinedemanating light and, in response, provide encoded combined emanatinglight at a second range of photon energies; a first filter locatedsubstantially adjacent to the first photo-sensor, wherein the firstfilter is configured to pass a first portion of the encoded combinedemanating light that corresponds to the first range of photon energiesto the first photo-sensor; and a second filter located substantiallyadjacent to the second photo-sensor, wherein the second filter isconfigured to pass a second portion of the encoded combined emanatinglight that corresponds to the second range of photon energies to thesecond photo-sensor.

According to the second aspect, the first photo-sensor is configured toreceive the filtered first portion of the encoded combined emanatinglight that corresponds to the first range of photon energies and detecttime variation resulting from relative movement between the one or moremask arrangements and the objects, the time variation includinginformation about the objects, and wherein the first photo-sensor isfurther configured to provide a first set of electrical signalsindicating one or more sensed time-varying waveforms to host structurecircuitry, and the second photo-sensor is configured to receive thefiltered second portion of the encoded combined emanating light thatcorresponds to the second range of photon energies and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a second set of electrical signals indicating oneor more sensed time-varying waveforms to host circuitry.

Still further according to the second aspect, the first and secondexcitation light sources includes one of a laser diode and lightemitting diode, and the host structure further comprises: an opticaldevice for re-directing encoded combined emanating light at either afirst range of photon energies or at a second range of photon energies.Still further according to the second aspect, the filter is furtherconfigured to receive and filter color-dependent encoded emanatinglight, and wherein the photo-sensor is further configured to receive thefiltered color-dependent encoded emanating light and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the photo-sensor is further configured toprovide an electrical signal indicating one or more sensed time-varyingwaveforms.

According to the second aspect, the host structure comprises: a firstexcitation light source; a second excitation light source; a firstfilter configured to receive and filter first color-dependent encodedemanating light provided by the fluid-engaging structure, the firstfilter located substantially adjacent to a first of the one or more maskarrangements located on the fluid-engaging structure; a second filterconfigured to receive and filter second color-dependent encodedemanating light provided by the fluid engaging structure, the secondfilter located substantially adjacent to a second of the one or moremask arrangements located on the fluid-engaging structure; a firstphoto-sensor located substantially adjacent to the first filter, andwherein the first photo-sensor is configured to receive the firstfiltered color-dependent encoded emanating light and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the first photo-sensor is furtherconfigured to provide a first electrical signal indicating one or moresensed time-varying waveforms; and a second photo-sensor locatedsubstantially adjacent to the second filter, and wherein the secondphoto-sensor is configured to receive the second filteredcolor-dependent encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a third electrical signal indicating one or moresensed time-varying waveforms, and wherein the circuitry is furtherconfigured to receive the first electrical signals and the thirdelectrical signals, and which is further configured to provide fourthelectrical signals in response to the first and third electrical signalsresulting from photosensing of the encoded emanating light, and whereinthe fourth electrical signals indicate the information about theobjects.

According to the second aspect, the host structure further comprises: aslot, wherein the slot is configured to receive the fluid-engagingstructure, to provide a substantially lossless light-interface between afirst excitation light source and the fluid engaging structure, and tofurther provide a substantially lossless light-interface between a firstphotosensor and the fluid engaging structure.

Still further according to the second aspect, the host structure furthercomprises: a second photosensor; and a second excitation light source,and wherein the slot is configured to provide a substantially losslesslight-interface between the second excitation light source and the fluidengaging structure, and to further provide a substantially losslesslight-interface between the second photosensor and the fluid engagingstructure, and the host structure further comprises: an interface boardconfigured to accept data entries by a user of the article ofmanufacture; and a display, wherein the display is configured to displaydata about the fluid to the user of the article of manufacture, andwherein the host structure is further configured to be one of either ahand-held unit, or an in-line unit.

According to the second aspect, a method of using the article ofmanufacture is provided comprising: receiving the fluid-engagingstructure, wherein the fluid engaging structure includes the fluid andobjects; determining information about the objects within the fluid; anddisplaying the information about the objects within the fluid to theuser of the article of manufacture. According to the second aspect, thestep of receiving the fluid-engaging structure comprises: locating thefluid-engaging structure in a slot in the host structure, wherein theslot is configured to secure temporarily the fluid-engaging structure tothe host structure, provide a first light-interface between a firstexcitation light source on the host structure and the fluid engagingstructure, and further provide a second light-interface between a firstphotosensor on the host structure and the fluid engaging structure, andwherein substantially no fluid in the fluid-engaging structure contactsthe host structure. According to the second aspect, the step ofdetermining information about the objects within the fluid comprises:receiving filtered encoded emanating light from the fluid-engagingstructure by a photo-sensor, wherein the encoded emanating lightincludes time variation resulting from relative movement between the oneor more mask arrangements of the fluid-engaging structure and theobject, the time variation including information about the object;detecting time variation resulting from relative movement between theone or more mask arrangements and the objects by the photo-sensor;providing a first electrical signal from the photo-sensor that indicatesone or more sensed time-varying waveforms; and receiving by hostcircuitry the first electrical signals; and providing second electricalsignals in response to the first electrical signals, wherein the secondelectrical signals indicate the information about the objects, and theinformation includes at least one of a type of the object, a quantity ofthe objects, a velocity of the objects, and a color of the objects.

A third aspect that overcomes all of the above described disadvantagesand provides a number of advantages includes an article of manufacturecomprising: a host structure, wherein the host structure includes afirst excitation light source; a second excitation light source; one ormore support parts structured to support a fluid-engaging structure onthe host structure, wherein the fluid-engaging structure includes achannel that in use can contain fluid and through which objects canmove; one or more bounding parts that bound the channel, and wherein atleast one of the one or more bounding parts include one or more lighttransmissive portions, wherein at least one of the one or more lighttransmissive portions is configured to receive excitation light andprovide the received excitation light, and wherein excitation lightenters the channel and interacts with an object resulting in emanatinglight; a first mask arrangement configured to receive at least part ofthe emanating light and, in response, provide encoded emanating light,and wherein the first filter arrangement and channel are furtherconfigured so that the encoded emanating light includes time variationresulting from relative movement between the first mask arrangement andthe object, the time variation including information about the object; afirst filter configured to receive and filter the encoded emanatinglight from the first mask arrangement; and a first photo-sensorconfigured to receive the filtered encoded emanating light from thefirst filter and detect time variation resulting from relative movementbetween the one or more mask arrangements and the object, the timevariation including information about the objects, and wherein the firstphoto-sensor is further configured to provide a first electrical signalindicating one or more sensed time-varying waveforms; a first electricalinterconnection; and a second mask arrangement configured to receive atleast part of the emanating light, and, in response, provide encodedemanating light, and wherein the second mask arrangement and channel arefurther configured so that the encoded emanating light includes timevariation resulting from relative movement between the second maskarrangement and the object, the time variation including informationabout the object; a second filter configured to receive and filter theencoded emanating light from the second filter arrangement; and a secondphoto-sensor configured to receive the filtered encoded emanating lightfrom the second filter and detect time variation resulting from relativemovement between the second mask arrangement and the object, the timevariation including information about the object, and wherein the secondphoto-sensor is further configured to provide a third electrical signalindicating one or more sensed time-varying waveforms; and circuitryconfigured to receive the first and third electrical signals and whichis further configured to provide fourth electrical signals in responseto the first and third electrical signals resulting from photosensing ofthe encoded emanating light, and wherein the fourth electrical signalsindicate the information about the object.

According to the third aspect, the host structure is configured for useas a point-of-care apparatus, the excitation light source includes oneof a light emitting diode and laser diode, and the at least onephoto-sensor is one of a large area photo sensor and a PIN diode.

These and other features and advantages of exemplary embodiments of theinvention are described below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing features of techniques in which afilter arrangement transmits and/or reflects light emanating from anobject with time variation.

FIG. 2 is a schematic diagram showing components of a system in whichlight emanating from an object can include information aboutcharacteristics of the object.

FIG. 3 is a schematic diagram of an excitation arrangement in anencoding component as in FIG. 2.

FIG. 4 is a schematic diagram of a filter arrangement in an encodingcomponent as in FIG. 2.

FIG. 5 is a schematic diagram of a displacement control arrangement inan encoding component as in FIG. 2.

FIG. 6 is a schematic block diagram of a system in which components asin FIG. 2 can be implemented.

FIG. 7 is a flow chart showing general operations in an implementationof an object distinguishing routine as in FIG. 6.

FIG. 8 is a schematic diagram of an analyzer in a fluidic structure,where the analyzer includes a system that can be implemented as in FIGS.6 and 7.

FIG. 9 is a top view of an article that can include a filter arrangementand that can be included in an encoding component as in FIG. 2.

FIG. 10 is a cross-sectional view of an implementation of an articlesimilar to that in FIG. 9, taken along the line 10-10.

FIG. 11 is a cross-sectional view of another implementation of anarticle similar to that in FIG. 9, taken along the line 11-11, togetherwith graphs of sensed intensities.

FIG. 12 is a partially schematic cross-sectional view showing two waysin which a filter arrangement on a photosensitive surface can beconfigured in an encoding component as in FIG. 2.

FIG. 13 is a schematic top view of another filter arrangement that canbe included in an encoding component as in FIG. 2.

FIG. 14 is a cross-sectional view of an implementation of a filterarrangement similar to that in FIG. 14, taken along the line 14-14,together with graphs of transmitted intensities.

FIG. 15 is a cross-sectional view of another implementation of a filterarrangement that can be included in an encoding component as in FIG. 2,together with graphs showing spectra of transmitted intensities.

FIG. 16 is a cross-sectional view of yet another implementation of afilter assembly that can be included in an encoding component as in FIG.2.

FIG. 17 is a cross-sectional view of yet another implementation of afilter assembly that can be included in an encoding component as in FIG.2.

FIG. 18 is a cross-sectional view of yet another implementation of afilter assembly that can be included in an encoding component as in FIG.2, such as with features as in any of FIGS. 15-17.

FIG. 19 is another partially schematic cross-sectional view showing adisplacement control arrangement that includes shaped boundaries,together with graphs showing velocity of an object and also showingintensity of emanating light as a function of time.

FIG. 20 is a cross-sectional view of another displacement controlarrangement that can be included in an encoding component as in FIG. 2,together with graphs showing intensity of emanating light for exemplarytypes of objects.

FIG. 21 is a partially schematic cross-sectional view of anotherdisplacement control arrangement that can be included in an encodingcomponent as in FIG. 2, together with a graph showing displacement as afunction of time and graphs showing intensity of emanating light as afunction of time for exemplary types of objects.

FIG. 22 is a top view of an implementation of a fluidic channel with anencoding arrangement that can be included in an implementation withfeatures as in FIG. 1.

FIG. 23 is a cross-sectional view of a component in FIG. 22, taken alongthe line 23-23.

FIG. 24 is a cross-sectional view of another component in FIG. 22, takenalong the line 24-24.

FIG. 25 includes a set of graphs showing cross-sectional thickness as afunction of position in an x-direction for filters and showingtransmission as a function of position in the x-direction or as afunction of time t for one of the filters.

FIG. 26 includes a set of graphs showing cross-sectional thickness as afunction of position in an x-direction for other filters and showingtransmission as a function of position in the x-direction or as afunction of time t for one of the filters.

FIG. 27 is a schematic cross-sectional view of a filter assembly thatincludes two simpler filters.

FIG. 28 is a flow chart with graphs illustrating an implementation inwhich information about objects is obtained from sensed time-varyingsignals.

FIG. 29 illustrates an embodiment of a fluidic chip for detection ofspatially modulated fluorescence along with an expected time-dependentoutput electrical signal according to an embodiment of the presentinvention.

FIG. 30 illustrates imposition of spatial modulation on fluorescenceemission from a moving particle for a conventional flow cytometer with ahighly focused spot of light.

FIG. 31 illustrates imposition of spatial modulation on a largerfluorescence emission than in FIG. 30 from a moving particle for aconventional flow cytometer with a lesser focused spot of light thanshown in FIG. 30.

FIG. 32 illustrates imposition of spatial modulation on fluorescenceemission from a moving particle with a patterned collection zonesuperimposed on the fluorescence emission for a flow cytometer accordingto an embodiment of the present invention.

FIG. 33 illustrates an alternate embodiment of a fluidic chip, withlaser excitation and apparatus to collect spatially modulatedfluorescence according to an embodiment of the present invention.

FIG. 34 illustrates a detailed top view of the fluidic chip and filtermask shown in FIG. 33 according to an embodiment of the presentinvention.

FIG. 35A illustrates an alternate embodiment of a fluidic chip, withlaser excitation and apparatus to collect spatially modulatedfluorescence according to an embodiment of the present invention.

FIG. 35B illustrates a detailed top view of the embodiment of the filtermask shown in FIG. 35A according to an embodiment of the presentinvention.

FIG. 36 illustrates a histogram of detected tagged cells as a functionof fluorescent intensity for a blood sample containing CD4 lymphocytesand CD4 monocytes from data obtained using an experimental set-upsubstantially similar to that as shown in FIGS. 35A and 35B according toan embodiment of the present invention.

FIG. 37 illustrates measured fluorescent amplitude for each detectedtagged cell as a function of particle speed for a blood samplecontaining CD4 lymphocytes and CD4 monocytes from data obtained using anexperimental set-up substantially similar to that as shown in FIGS. 35Aand 35B according to an embodiment of the present invention.

FIGS. 38-40 illustrate several screenshots of data acquisition in theexperimental set-up substantially similar to that as shown in FIGS. 35Aand 35B that illustrate the presence of two closely-spaced CD4 cells andtheir respective speeds.

FIG. 41 illustrates a pattern for the major constituents of white bloodcells obtained from whole blood obtained with a known reagent from dataobtained using an experimental set-up substantially similar to that asshown in FIGS. 35A and 35B according to an embodiment of the presentinvention.

FIG. 42 illustrates a cut-away side view of an alternate embodiment of afluidic-chip and part of a host-structure according to an alternateembodiment of the present invention.

FIG. 43 illustrates a side view of a prototype flow cytometer with thealternate embodiment of the fluidic chip for detection of spatiallymodulated fluorescence shown in FIG. 42 according to an embodiment ofthe present invention.

FIG. 44 illustrates a front perspective view of the prototype flowcytometer and fluidic chip as shown in FIG. 43.

FIG. 45 illustrates a top perspective view of the fluidic chip fordetection of spatially modulated fluorescence as shown in FIG. 42.

FIG. 46 illustrates a cut-away side view of the fluidic chip shown inFIGS. 42 and 45 and a portion of the prototype flow cytometer shown inFIGS. 43 and 44 according to an embodiment of the present invention.

FIG. 47 illustrates an intensity histogram of a sample containingcalibration beads from data obtained using the prototype flow cytometershown in FIGS. 43 and 44.

FIG. 48 illustrates a block diagram of the prototype flow cytometershown in FIGS. 43 and 44 with an additional light source according to anembodiment of the present invention.

FIG. 49 illustrates a cut-away side view of an alternate embodiment of afluidic-chip and part of a host-structure according to an alternateembodiment of the present invention.

FIG. 50 illustrates a cut-away side view of an alternate embodiment of afluidic-chip and part of a host-structure according to an alternateembodiment of the present invention.

FIG. 51 illustrates a front perspective view of a commercial embodimentof a disposable fluidic-chip, and a handheld host-structure implementedas a point-of-care device for use with the disposable fluidic chipaccording to an embodiment of the present invention.

FIG. 52 is a partial cut-away top view of the point-of-care flowcytometer and disposable fluidic chip as shown in FIG. 51.

FIG. 53 is a partial cut-away side view of the point-of-care flowcytometer and disposable fluidic chip as shown in FIG. 51.

FIG. 54 illustrates a cut-away side view of an alternate embodiment ofthe fluidic-chip as shown in FIG. 45 with a sample and waste reservoirintegrated within the fluidic-chip, according to an embodiment of thepresent invention.

FIG. 55 illustrates a cut-away side view of alternate embodiment of afluidic-chip and part of a host-structure according to an alternateembodiment of the present invention.

FIG. 56 illustrates a cut-away side view of alternate embodiment of afluidic-chip and part of a host-structure according to an alternateembodiment of the present invention.

FIG. 57 illustrates a cut-away side view of alternate embodiment of afluidic-chip and part of a host-structure according to an alternateembodiment of the present invention.

FIG. 58 illustrates a top view of a filter mask and channel of a fluidicchip indicating alignment along a centerline of the channel according toan embodiment of the present invention.

FIG. 59 illustrates a side view of a filter mask and channel of afluidic chip indicating alignment along an upper boundary portion of thechannel according to an embodiment of the present invention.

FIG. 60 illustrates a front view of a filter mask and channel of afluidic chip in the direction of flow of the fluid indicating alignmentalong an upper boundary portion of the channel according to anembodiment of the present invention.

FIG. 61 illustrates a cut-away side view of alternate embodiment of afluidic-chip and part of a host-structure according to an alternateembodiment of the present invention.

FIG. 62 illustrates a top view of a patterned colored maskfilter-arrangement used in the fluidic-chip shown in FIG. 61.

FIG. 63 illustrates complementary time-dependent, orthogonal detectorsignals of a red emitting particle and a green emitting particleaccording to an embodiment of the present invention.

DETAILED DESCRIPTION

In the following detailed description, numeric values and ranges areprovided for various aspects of the implementations described. Thesevalues and ranges are to be treated as examples only, and are notintended to limit the scope of the claims. In addition, a number ofmaterials are identified as suitable for various facets of theimplementations. These materials are to be treated as exemplary, and arenot intended to limit the scope of the claims.

“Light” refers herein to electromagnetic radiation of any wavelength orfrequency; unless otherwise indicated, a specific value for lightwavelength or frequency is that of light propagating through vacuum.Light that can include information is sometimes referred to herein as an“optical signal”.

The term “sensing” is used herein in the most generic sense of obtaininginformation from a physical stimulus; sensing therefore includes actionssuch as detecting, measuring, and so forth. A “sensor” is a device thatperforms sensing. Data or other signals that indicate or include resultsof sensing are sometimes referred to herein as “sensing results”.

“Photosensing” is sensing of light. A “photosensor” is accordingly anelectronic device that performs photosensing. More specifically, ifoptical signals include information, a photosensor that receives theoptical signals may be able to sense the information and provide sensingresults that indicate or include the information. A surface at whichphotosensing occurs is referred to herein as a “photosensitive surface”.

The various exemplary implementations described below address problemsthat arise in obtaining information about a moving object such as abiological cell, a virus, a molecule, or a submolecular complex, such asin flow cytometry. Flow cytometry has become an indispensable tool inclinical diagnostics, such as in diagnosing cancer, AIDS, and infectiousdiseases during outbreaks, and also in microbiology and other areas. Thecost and size of existing cytometers preclude their use in fieldclinics, water monitoring, agriculture/veterinary diagnostics, andrapidly deployable biothreat detection.

A number of commercially available flow cytometers use multipleexcitation sources, each focused on a well-defined location or regionseparate from the others. Light emitted from each source's region istypically analyzed with a series of beam splitters, filters, andphotomultiplier tubes (PMTs) in order to detect and distinguishdifferently stained cells or cells that concurrently carry multipledyes. Cells are typically stained in solution with different dyes priorto insertion into a cytometer, and the measurement takes place in afluidic channel in which cells traverse a detection region, typically ata speed of up to several meters per second. In the detection region,focused laser light (typically with an elliptical focus of 80 μm×40 μm)excites the dyes on the cells. The resulting fluorescent light can becollected by a microscope lens, sorted by band pass filters, anddetected by PMTs or avalanche photodiodes (APDs). For each spotexcitation, a respective set of filters and detectors is needed, whichis costly and leads to bulky devices with strict requirements necessaryto maintain optical alignment. Since the detection region is small andobjects traverse it rapidly (typical dwell times are around 10 μsec),such flow cytometers have serious signal-to-noise (S/N) ratio issues forweakly fluorescing cells. These issues become more acute if multipletargets must be characterized and distinguished in some way, such as bycounting.

A major cost in flow cytometry applied in clinical diagnostics is costof reagents (e.g. antibodies and conjugated dyes). There are two ways toreduce the amount of consumables: First, one can reduce the requiredamount of analyte, e.g. by employing microfluidic techniques; andsecond, one can reduce the amount of consumable per analyte volume.Reducing amounts used would, however, reduce fluorescent intensity. Itwould be valuable to be able to overcome this constraint with acost-effective and reliable technique to detect and distinguish weaklyemitting cells.

Previous proposals to address these problems have involved spatiallymodulated single-color excitation to improve S/N ratios and to shift thedetection limit toward weaker emitting cells. Spatial resolution can bemaintained or improved in comparison with previous flow cytometrytechniques, because fluorescing light is spatially modulated over acomparably large detection region; this is helpful because spatialresolution affects maximum detection or count rate of a device. Butsingle-color techniques are limited, whether excitation is performed ina black/white approach or with a single-color interference pattern froma light source. Also, single-color techniques can encounter problemswith wavelength sensitivity and bleaching of dyes. Because of lowwavelength sensitivity, many flow cytometers with filter-PMTcombinations are also constrained to use dyes with substantiallydifferent fluorescence wavelengths.

In addressing such problems, some exemplary implementations describedbelow employ filter arrangements that transmit or reflect emanatinglight with one or both of two techniques: A filter assembly is used thatprovides different transmission functions in different segments of anobject's path and/or a filter component is used that has a combinedtransmission function in which a set of simpler non-uniform transmissionfunctions are superimposed. Such techniques make it possible to provideseveral different transmission functions in sequence within relativelyshort part of an object's path, so that the object's emanating light isrelatively constant across the different transmission functions. Thesetechniques also allow much greater variation in filter arrangements thanwould be possible with binary, black/white masks or single color masks.In addition, these techniques can be implemented to maintain higherspatial resolution and to allow higher photon flux on a photosensor.Time variation of emanating light resulting from such filters mayprovide sufficient information to make spectral characterization ofparticles feasible. Use of multiple colors may be compatible withparticle identification based on native fluorescence; in particular,patterned filter arrangements allow for detection of differences inemission spectra and even the very small differences that occur innative fluorescence spectra might be detectable. It may also enableadvanced color monitoring in printing applications by detecting evensmall differences in the reflection spectra of color spots while theyare moving past interdigitated or otherwise patchworked or patternedfilter arrangements.

The term “photon” refers herein to a quantum of light, and the term“photon energy” refers herein to the energy of a photon. Light can bedescribed as having a “photon energy distribution” or, more commonly, a“spectrum”, meaning the combination of photon energies that are includedin the light; highly monochromatic light, for example, has a photonenergy distribution or spectrum with one peak energy value.

Light can also be described as provided by a “light source,” which,unless otherwise specified, refers herein to any device, component, orstructure that can provide light of the type described; examples oflight sources relevant to the below-described implementations includevarious kinds of pulsed and unpulsed lasers and laser structures, laserdiodes (LDs), light emitting diodes (LEDs), superluminescent LEDs(SLEDs), resonant cavity LEDs, sources of broadband light that isspectrally filtered such as with a monochromator, and so forth.

To “propagate” light through a region or structure is to transmit orotherwise cause the light to propagate through the region or structure.The light may be referred to as “propagated light” or “propagatinglight”.

Propagating light can often be usefully characterized by direction andspeed of propagation, with direction typically illustrated by one ormore rays and with speed typically being described relative to theconstant c, also referred to as the speed of light in vacuum. Where thespeed of light in a medium M is a constant c_(M) less than c, then M hasan index of refraction n_(M)=c/c_(M).

Where light changes direction in a way that can be illustrated orapproximated as a vertex between an incoming ray and an outgoing raythat are both on one side of a surface, the change may be referred to asa “reflection”; similarly, to “reflect” light is to cause the light tochange its direction of propagation approximately at such a surface,referred to herein as a “reflection surface”. Similarly, where lightchanges direction in a way that can be illustrated or approximated as avertex between an incoming ray and an outgoing ray that are on oppositesides of a surface between two media with different indices ofrefraction, the change may be referred to as a “refraction”; similarly,to “refract” light is to cause the light to change its direction ofpropagation approximately at such a surface, referred to herein as a“refraction surface”. In many practical applications, both reflectionand refraction occur at a surface, which may be referred to herein as a“partially reflecting surface”.

Where light propagates at less than c, it may be useful to obtain an“optical distance” of propagation; for any segment of length d in whichspeed of propagation is constant ε*c, where ε=1/n_(EFF)≦1 and n_(EFF) isan effective index of refraction for the segment, optical distanceD(ε)=d/ε. An optical distance may be referred to herein as an “opticalthickness”, such as where light is propagating through a thickness ofmaterial.

A “range of photon energies” or an “energy range” is a range of energyvalues that photons can have. An energy range can be described, forexample, as a range of wavelengths or a range of frequencies or, inappropriate cases, by the range's central wavelength or frequency andpossibly also the range's width. A “subrange” of a range of photonenergies is a part of the range, and can be similarly described.

In general, the upper and lower boundaries and widths of ranges andsubranges are approximate. To provide output photons or to photosensequantity of photons “throughout”, “within”, or “in” a range or subrangemeans to provide photons or to obtain information about quantity ofphotons that are predominantly within the range or subrange. In typicalcases, between 60-90% of the provided photons or sensed quantity ofphotons have energies within the range or subrange, but the percentagecould be lower or higher. In some applications, 90% or even 95% or moreof the provided photons or sensed quantity of photons have energieswithin the range or subrange.

The term “electrical signal” is used herein to encompass any signal thattransfers information from one position or region to another in anelectrical, electronic, electromagnetic, or magnetic form. Electricalsignals may be conducted from one position or region to another byelectrical or magnetic conductors, but the broad scope of electricalsignals also includes light and other electromagnetic forms of signalsand other signals transferred through non-conductive regions due toelectrical, electronic, electromagnetic, or magnetic effects. Ingeneral, the broad category of electrical signals includes both “analog”and “digital” signals: An “analog” electrical signal includesinformation in the form of a continuously variable physical quantity,such as voltage; a “digital” electrical signal, in contrast, includesinformation in the form of discrete values of a physical characteristic,which could also be, for example, voltage.

Some implementations of filter arrangements described herein employstructures with one or more dimensions smaller than 1 mm, and varioustechniques have been proposed for producing such structures. Inparticular, some techniques for producing such structures are referredto as “microfabrication.” Examples of microfabrication include varioustechniques for depositing materials such as growth of epitaxialmaterial, sputter deposition, evaporation techniques, platingtechniques, spin coating, printing, and other such techniques;techniques for patterning materials, such as etching or otherwiseremoving exposed regions of thin films through a photolithographicallypatterned resist layer or other patterned layer; techniques forpolishing, planarizing, or otherwise modifying exposed surfaces ofmaterials; and so forth.

In the implementations described below, structures, systems, or parts orcomponents of structures or systems may sometimes be referred to as“attached” to each other or to other structures, systems, parts, orcomponents or visa versa, and operations are performed that “attach”structures, systems, or parts or components of structures or systems toeach other or to other things or visa versa; the terms “attached”,“attach”, and related terms refer to any type of connecting that couldbe performed in the context. One type of attaching is “mounting”, whichoccurs when a first part or component is attached to a second part orcomponent that functions as a support for the first. In contrast, themore generic term “connecting” includes not only “attaching” and“mounting”, but also making other types of connections such aselectrical connections between or among devices or components ofcircuitry. A combination of one or more parts connected in any way issometimes referred to herein as a “structure”.

Some of the structures, elements, and components described herein aresupported on a “support structure” or “support surface”, which terms areused herein to mean a structure or a structure's surface that cansupport other structures. More specifically, a support structure couldbe a “substrate”, used herein to mean a support structure on a surfaceof which other structures can be formed or attached by microfabricationor similar processes.

The surface of a substrate or other support surface is treated herein asproviding a directional orientation as follows: A direction away fromthe surface is “up”, “over”, or “above”, while a direction toward thesurface is “down”, “under”, or “below”. The terms “upper” and “top” aretypically applied to structures, components, or surfaces disposed awayfrom the surface, while “lower” or “underlying” are applied tostructures, components, or surfaces disposed toward the surface. Ingeneral, it should be understood that the above directional orientationis arbitrary and only for ease of description, and that a supportstructure or substrate may have any appropriate orientation.

A structure may be described by its operation, such as a “supportstructure” that can operate as a support as described above; otherexamples are defined below. In addition, a structure may becharacterized by the nature of its parts or the way in which they areconnected; for example, a “layered structure” is a structure thatincludes one or more layers, and the terms “partial structure” and“substructure” refer to structures that are in turn parts of otherstructures.

Unless the context indicates otherwise, the terms “circuitry” and“circuit” are used herein to refer to structures in which one or moreelectronic components have sufficient electrical connections to operatetogether or in a related manner. In some instances, an item of circuitrycan include more than one circuit. An item of circuitry that includes a“processor” may sometimes be analyzed into “hardware” and “software”components; in this context, “software” refers to stored or transmitteddata that controls operation of the processor or that is accessed by theprocessor while operating, and “hardware” refers to components thatstore, transmit, and operate on the data. The distinction between“software” and “hardware” is not always clear-cut, however, because somecomponents share characteristics of both; also, a given softwarecomponent can often be replaced by an equivalent hardware componentwithout significantly changing operation of circuitry, and a givenhardware component can similarly be replaced by equivalent processoroperations controlled by software.

Circuitry can be described based on its operation or othercharacteristics. For example, circuitry that performs control operationsis sometimes referred to herein as “control circuitry” and circuitrythat performs processing operations is sometimes referred to herein as“processing circuitry”.

In general, sensors, processors, and other such items may be included ina system in which they are operated automatically or partiallyautomatically. As used herein, the term “system” refers to a combinationof two or more parts or components that can perform an operationtogether. A system may be characterized by its operation; for example,an “object distinguishing system” is a system that operates somehow todistinguish objects.

Within a system, device, or other article, components and parts may bereferred to in a similar manner. One component of an objectdistinguishing system, for example, can be described as an “encodingcomponent”, in some cases referred to as an “encoding arrangement”, ineither case meaning that the component or arrangement operates to encodeinformation; similarly, a system can include an “filter component”, insome cases referred to as an “filter arrangement”, in either casemeaning that the component or arrangement operates to perform filtering,as explained in greater detail below; various other components aredescribed below. In addition, a component or part may be identified bycharacteristics other than its operation.

In FIG. 1, object 10 is one of series 12 of objects 14 through 16 thattravel along respective paths past filter arrangement 20. The term“path” is used herein in the general sense of a series of positionsand/or configurations that a moving and/or varying object can haveduring its motion and/or variation. For generality, a part of a path issometimes referred to herein as a “segment”, which could encompass anycontinuous series of one or more positions and/or configurations withina path.

As object 10 travels past arrangement 20, light emanates from it, suchas by emission, scattering (including, e.g. reflection), ortransmission, and a portion of the emanating light is received by filterarrangement 20, as indicated by arrow 22. In general, the emanatinglight includes light within an application's range of photon energies,meaning that techniques as in FIG. 1 can be successfully used in a givenapplication, e.g. flow cytometry, bio-chip readout, or any suitable kindof analyte detection, even though emanating light might also includephoton energies that are outside the application's range and that mightnot interact with filter arrangement 20 in the same way as light in theapplication's range.

The term “object” is used herein in the general sense of anydistinguishable thing about which information can be obtained by asensor and included in its sensing results. In some implementations,sensors can obtain information about objects by receiving signals fromthem; for example, signals in the form of light can emanate from anobject, whether through emission (e.g. radiation, fluorescence,incandescence, chemoluminescence, bioluminescence, other forms ofluminescence, etc.), scattering (e.g. reflection, deflection,diffraction, refraction, etc.), or transmission, and can be sensed by aphotosensor. The light “emanates from” or is simply “from” the object,and may be referred to herein as “emanating light”. An object from whichlight is emanating may be referred to herein as a “light-emanatingobject”. In other implementations, sensors can obtain information aboutobjects in other ways, some of which are mentioned herein.

Examples of objects that could occur in implementations as describedbelow include droplets, small volumes of fluid, single molecules,agglomerated molecules, molecule clusters, cells, viruses, bacteria,lengthy polymers such as DNA or protein chains, submolecular complexessuch as tags on DNA or protein chains, microparticles, nanoparticles,beads or other small particles that can bind and carry specificchemicals or other analytes, emulsions, any such type of object in anarray such as an array of sample wells, and a distinguishable region ofa surface such as a small area of a sheet of paper or otherimage-bearing medium; a distinguishable region, could, for example, be acolored spot. A droplet or small volume of fluid may, for example,include atoms, molecules, or other particles that emit lightspontaneously or in response to excitation; a particle could be a“fluorescent component” of a droplet, fluorescing in response toexcitation. Or a droplet may include particles that absorb lightincident on the droplet, so that the droplet does not reflect orotherwise scatter the absorbed light; in this case, a particle could bean “absorbent component” of a droplet. Or a droplet may includeparticles that scatter light incident on the droplet in a way thatdepends on photon energy, so that the droplet scatters the incidentlight correspondingly; in this case, a particle could be a “scatteringcomponent” of a droplet. An analyte (i.e. a chemical species beinginvestigated) in a droplet or other object can act as a fluorescent,absorbent, or scattering component. Analyte that is otherwisehomogeneously distributed, for example, can be localized by binding tocarrier beads, resulting in a moving object that emanates light orprovides other signals in a way that depends on the analyte.

With respect to a light-emanating object, the expressions“characteristic of an object” and “emanating light includinginformation” have related meanings: The term “characteristic” refers toa trait, quality, or property of an object that can be measured and thatpersists with a given value or within a given range or other subset ofpossible values while light that “includes information about thecharacteristic” is emanating from the object. In appropriateimplementations, characteristics of an object could include mass,volume, density, cross-section or other shape, chemical composition,position, speed, acceleration, direction of movement, spin axis,directional or angular velocity or momentum, net charge, chargepolarity, absorption spectrum, emission spectrum, scattering spectrum,and so forth. Therefore, emanating light “includes” information about acharacteristic of an object if information included in the emanatinglight indicates a value, range, or other measure of the characteristic.Similar terminology can apply to types of signals other than emanatinglight.

Emanating light or other types of signals can “include information” inmany ways, some of which are described below in relation to specificimplementations. Various criteria could be used to determine whetheremanating light or another type of signal includes specifiedinformation, and such criteria can be referred to as “encodingcriteria”. Some encoding criteria, for example, involve comparison ofmagnitude of a signal with noise magnitude, e.g. signal-to-noise (S/N)ratios, because S/N ratio can affect whether specified information canbe recovered from sensing results obtained by photosensing emanatinglight. Other types of encoding criteria could be used as appropriate.Where emanating light or another type of signal satisfies an appropriateencoding criterion for specified information, the light or signal may besaid to “encode” the information.

Similarly, sensing results, whether from photosensing emanating light orfrom another type of sensing, can “include information” in many ways,and similar encoding criteria could be applied as with signals. Wheresensing results indicate one or more time-varying waveforms, the sensingresults can be referred to as having “encoded time variation”.

The term “waveform” is used herein in the general sense of any set ofvalues that varies over one or more dimensions, whether continuous ordiscrete, whether analog or digital, and whether measured or obtained inany other way; a “time-varying waveform” is a waveform that varies overa time dimension. Some of the time-varying waveforms described below inrelation to exemplary implementations include intensity values, but theexpression “time-varying waveforms” also encompasses other values thatvary over time, including purely numerical values with no specifiedunits or other physical significance. A “sensed time-varying waveform”is a time-varying waveform that is indicated by sensing results obtainedover time. For example, if a photosensor provides sensed quantities thatindicate intensity of received light, its sensing results could indicatea time-varying waveform indicating intensity sensed over time.

In a system in which sensing results, emanating light, or other signalscan include information about characteristics of objects, an object“travels” or is caused “to travel” if the object has a succession ofpositions over time with respect to one or more parts or components ofthe system or one or more patterns or other features of the system'senvironment such that information about the object's traveling, e.g.about speed or other rate of displacement, can be included in theemanating light or other signals. An object that travels is sometimesalso referred to herein as “moving” or as having “motion” or “movement”,but an object's traveling may result from any appropriate motion of theobject and/or motion of parts or components of the system or patterns orother features of its environment. In other words, motion of an objectincludes any relative movement between the object and parts orcomponents of a system or patterns or features of the system'senvironment, such as an encoding or sensing component of the system or apattern of excitation or of filtering or another environmental patternor feature.

A moving object's path is treated herein as providing a directionalorientation as follows: A direction parallel or approximately parallelto the path is sometimes referred to as a “longitudinal” or “lengthwise”direction, while a direction perpendicular or approximatelyperpendicular to the path is sometimes referred to as a “radial”,“lateral”, or “transverse” direction. The lengthwise direction in whichthe object is moving is sometimes referred to as “forward” or“downstream”, while the opposite direction is sometimes referred to as“backward” or “upstream”. A radial direction away from the path is “out”or “outward”, while a radial direction toward the path is “in” or“inward”. Light propagating toward the path may be referred to as“incoming” or “incident”, while light propagating away from the path maybe referred to as “outgoing”. A component or arrangement of componentsis “along” the path if it is disposed near the path and has some extentin a longitudinal direction. A component or arrangement of components is“around” the path if, in a plane transverse to the path, it intersectsmultiple radial directions, at least two of which are separated byapproximately 180 degrees of arc. A direction that similarly goes aroundthe path is sometimes referred to herein as a “rotation” direction. Ingeneral, it should be understood that the above directional orientationis arbitrary and only for ease of description, and that a movingobject's path may have any appropriate orientation.

Emanating light that includes information about an object's traveling issometimes referred to herein as “motion-affected” light, as including“motion-dependent information”, or as having “motion-dependentencoding”. For example, an object could travel by being conveyed influid, such as liquid, gas, or aerosol, along a path in which itemanates light that is transmitted and/or reflected by a filterarrangement to include information about the object's motion, thusbecoming motion-affected light; in such a case the object may bereferred to as being “carried” by fluid. In another example, an objectcontained in or otherwise supported by a support structure could traveldue to relative scanning movement between the support structure and afilter component or another component such as a photosensor, and itcould emanate light that is transmitted and/or reflected so that itbecomes motion-affected light.

The term “optical filter” or simply “filter” refers herein to alight-transmissive or light-reflective part or component that transmitsand/or reflects light in accordance with a respective criterion,sometimes referred to herein as a filter's “type”. For example, onegeneral category of filters is “band pass filters”, referring to typesof filters that, across some application's range of photon energies,e.g. a range of wavelengths or frequencies such as the visible range,preferentially transmit and/or reflect light within a subrange,sometimes referred to as a “band”; a band pass filter's type cantherefore be specified by specifying the band or subrange of photonenergies in which it transmits and/or reflects. A “blocking filter”,which does not transmit or reflect any light in an application's range,can be viewed as a band pass filter with a band of zero bandwidth, whilea “transparent filter”, which transmits and/or reflects all light in anapplication's range, can be viewed as a band pass filter with a bandthat includes the entire range.

Filters can be combined and configured in many different ways, and allsuch combinations and configurations of one or more filters areencompassed herein by the general term “filter arrangement”. A filterarrangement can include, for example, one or more “filter components”,one or more “filter assemblies”, and/or one or more “filter elements”;while the term “filter component” is generic, referring to any componentthat operates as a filter, the terms “filter assembly” and “filterelement” are related and therefore a bit more specific, in that a filterassembly is a filter component that includes one or more filterelements, while a filter element is a filter component that generallydoes not include other filter elements within it. In general, filterelements and filter assemblies are sometimes also referred to as“masks”. Also, the terms “transmit” and “reflect” and related words, asused herein, include each other unless otherwise specified, and termssuch as “transmit/reflect” or “transmitting/reflecting” encompasstransmission without reflection, reflection without transmission, andconcurrent transmission and reflection.

Filter elements of various kinds could be included in filter assemblies,filter components, filter arrangements, and other combinations andconfigurations of filters, in a wide variety of ways. Within a givenconfiguration of filters, relationships between filters can be describedin a number of ways. For example, light can pass through a “sequence” offilters, meaning that specified light passes through the filters in asequence: If a “radial sequence” of filters is along a path, forexample, emanating light can pass through each of the filters in thesequence, beginning with the first and, after passing through eachpreceding filter, passing through the following filter; of course, lightthat is blocked by a preceding filter in a radial sequence would notreach its following filter. If a “longitudinal sequence” of filters isalong a path, on the other hand, light emanating at each of a sequenceof segments of on the path passes through a respective filter in thelongitudinal sequence.

Several other categories of filters are described below in relation toexemplary implementations, including shadow masks, periodic masks, chirpmasks, random masks, and so forth, and various other categories could beidentified. As used herein, the term “random” refers to a pattern thatis non-periodic over the entire length of a longitudinal sequence offilters; in contrast, a “periodic” filter assembly has at least onepattern that repeats more than once across the assembly's longitudinallength; and “chirp” patterns meet the above definition of random butcan, with linearly varying time scaling, meet the above definition ofperiodic, in effect being a sequence of periods of linearly changingfrequency or wavelength. A “shadow mask” is not a band pass filter, butrather an intensity-based filter assembly that, within a photon energyrange of interest, transmits/reflects light of all energies, but withdifferent parts of the filter transmitting/reflecting the light atdifferent intensities, such as black and white and/or different grayscales. Any of these types of filter assemblies can be used to obtain“spatially modulated” emanating light, meaning emanating light thatvaries in time depending on position of an object from which it isemanating.

As used herein, the term “white”, in a given implementation, refers tolight with a spectrum that approximates maximum intensities across theimplementation's full range of photon energies (which could be broadband, a combination of red, green, and blue, or another appropriatecombination); the term “black” refers to the opposite of white, i.e.minimum available intensities across the full range, with the idealbeing no light and therefore zero intensities. In emanating spectra, forexample, light without maximal intensities across the full range as inwhite may be characterized as having a “gray level”, such as one of thegray levels between white and black, or as having a “color”, such as ifit includes predominantly photon energies in a subrange, e.g. one of thecolors in the visible range of wavelengths or in the infrared orultraviolet ranges, or possibly a combination of such colors. Spectrathat are neither black nor white are sometimes referred to herein as“non-binary spectra”. Filter arrangement 20 includes either or both oftwo specified combinations or configurations of filters: Filtercomponent 30 includes filter assembly 32 in which positions 34 and 36have different transmission functions as indicated by differentcross-hatching, while filter component 40 has a combined transmissionfunction, represented by box 42, in which two or more differentnon-uniform transmission functions are superimposed. As a result of thedifferences in transmission functions, information can be encoded intime variation of emanating light from objects such as object 10.

As used herein, the term “transmission function” refers to a functionthat indicates, for some appropriate position or set of positions, therelationship of output and input light of a light-transmissive and/orlight-reflective component such as a filter or filter assembly. Aposition's transmission function could indicate, for example, ratio ofoutput intensity to input intensity at the position across a range ofphoton energies, sometimes referred to herein as the transmissionfunction's “transmission spectrum”; a band pass filter, for example,could have approximately the same transmission spectrum at substantiallyall of its positions. A position could have any of a variety of otherkinds of transmission functions, including, for example, an “intensityratio”, indicating the ratio of the position's output intensity to itsinput intensity, where the same intensity ratio applies to all photonenergies across the relevant range; in the simple case in which eachposition of a filter has either an intensity ratio of zero or one, eachposition's transmission function could be one of a pair of binaryvalues, such as black/white, ON/OFF, one/zero, or the like.

Further, a band pass filter or other filter element or assembly has a“uniform transmission function” if substantially all its positions havetransmission functions that are approximately the same, and such atransmission function may be said to be “approximately uniform” forlight transmitted/reflected through the filter element or assembly.Conversely, a filter element or assembly has a “non-uniform transmissionfunction” if its transmission function is not approximately uniform;examples include periodic, random, and chirp filters as described above.

Transmission functions can, of course, be different from each other invarious ways. For example, transmission functions of two positions candiffer in “color”, meaning that the positions have differenttransmission spectra; transmission functions with transmission spectrathat have the same shape across a relevant range can differ in“intensity”, meaning that they have different intensity ratios. Similarterminology can be applied to uniform transmission functions for filterelements, components, or assemblies. In FIG. 1, elements 34 and 36 havetransmission functions that are different from each other. Also, simplertransmission functions that are superimposed to provide a combinedtransmission function can have different transmission functions; in FIG.1, two of the simpler transmission functions superimposed to providecombined transmission function 42 are non-uniform and different fromeach other.

Different transmission functions can also be combined in a number ofways. For example, in a longitudinal sequence of filters, transmissionfunctions are similarly combined into a sequence. In a stack or otherradial sequence of filters or filter assemblies, on the other hand,transmission functions can be “superimposed”, meaning that bothtransmission functions are applied to light passing through thecomponent, resulting in a combined transmission function in whichsimpler transmission functions are superimposed. As used herein, atransmission function is “simpler” than a combined transmission functionin which it is superimposed with at least one other transmissionfunction, except in cases where the combined transmission function andall of the superimposed transmission functions have the same spectralshape or where the superimposed transmission functions have relatedshapes that result in uniform loss of detail when superimposed inspecific phase relationships; although there are many abstract examplesof superpositions that result in uniform loss of detail (e.g. two squarewaves of the same period and at opposite phase would have a flat linesuperposition) simplifying superpositions are very unlikely to occurbetween transmission functions with disparate shapes, such as random andperiodic, random and chirped, chirped and periodic, and so forth--somedetail might be lost locally in such cases, but most detail ispreserved. Simpler transmission functions can be superimposed to obtaina combined transmission function in various ways other than a stack orradial sequence; for example, as described below in relation to someexemplary implementations, a single filter assembly can have a combinedtransmission function that is “stack-equivalent”, meaning that it isapproximately equivalent to a stack of filter components with simplertransmission functions. In some cases, including certain types ofreflective filters, a stack-equivalent filter assembly can be equivalentto a combination of simpler filters without regard to the order in whichthey are superimposed, so that it is equivalent to a number of differentstacks in which the simpler filters are in different orders.

As shown within combination 30, when object 10 is in segment 50 orsegment 52 of its path, respective portions of emanating light aretransmitted/reflected through positions 34 and 36 of filter assembly 32,as indicated respectively by arrows 54 and 56. Because of the differenttransmission functions, this transmitting/reflecting operation encodesinformation in time variation of the emanating light, represented byarrows 58. Specifically, if the emanating light from segments 50 and 52has the same intensity or spectrum, the output light from positions 34and 36 can affect its intensity or spectrum differently, and thisdifference can indicate, for example, the time at which object 10 movedbetween segment 50 and segment 52. In the illustrated example, segment50 precedes segment 52 along the path of object 10, so that the portionof emanating light from segment 50 is transmitted/reflected according tothe transmission function of position 34 before the portion of emanatinglight from segment 52 is transmitted/reflected according to thetransmission function of position 36.

In some exemplary implementations below, for example, a filter assemblycan have a longitudinal sequence of band pass filter elements with bandsof different colors. As a result, output light from filter elements ofdifferent colors will have different intensities, depending on thespectrum of light emanating from an object, so that time variation ofthe output light encodes information about the emanating light'sspectrum, i.e. about the type of the object. In other examples,information about speed or other displacement rate and position can beencoded by longitudinal filter sequences.

As shown within component 40, on the other hand, as object 10 passesthrough each of a series of segments that includes segments 60 and 62,respective portions of emanating light are transmitted/reflected withcombined transmission function 42, as indicated by respective arrows 64and 66. Because at least two of the simpler transmission functions thatare superimposed in function 42 are non-uniform and different from eachother, this transmitting/reflecting operation also encodes informationin time variation of the emanating light, represented by arrows 68. Ifthe emanating light from each segment in the series has the sameintensity or spectrum, the output light from function 42 will be encodedin accordance with both of the simpler non-uniform transmissionfunctions. In other words, information in accordance with both of thesimpler transmission functions will be concurrently encoded in timevariation of the emanating light.

In some exemplary implementations below, for example, a stack orstack-equivalent filter assembly combines a periodic or chirptransmission function that can encode information about an object'sposition, speed, or other displacement rate with a random transmissionfunction that can encode information about an object's spectrum or type.Emanating light passing through the filter assembly is concurrentlyencoded with both types of information.

As suggested by the words “AND/OR” between combination 30 and component40, the two are not mutually exclusive, and could be implementedtogether. As described below in relation to some exemplaryimplementations, a single filter assembly could encode information intime variation of emanating light in both of the ways illustrated forcombination 30 and component 40.

The operation in box 70 photosenses the emanating light that hasinformation encoded in its time variation, represented by arrows 58 and68. This operation can be implemented with any suitable photosensingcomponent, some of which are described below. In general, sensingresults from photosensing take the form of analog or digital electricalsignals, depending on the structure and circuitry included in thephotosensing component. The operation in box 72 uses the sensing resultsfrom box 70 to obtain data indicating some or all of the encodedinformation, and can therefore be referred to as a “decoding” operation.The results of decoding can be used in a wide variety of ways, some ofwhich are described below in relation to specific implementations.

Information about an object, as obtained in FIG. 1, can be used for awide variety of purposes. In exemplary implementations described below,such information can, for example, be used to distinguish objects. Insome applications, such as where the distinguished objects areregistration marks in documents or other images, appropriate subsequentoperations can be controlled based on the results of distinguishingobjects.

Filtering arrangement 20 in FIG. 1 could be implemented in manydifferent ways, some of which are described below. In some exemplaryimplementations below, for example, a filter component includespositions that have different transmission functions. In others, afilter assembly has a combined transmission function with superimposedsimpler, non-uniform transmission functions. These techniques can beimplemented together. As a result of these techniques, emanating lightwill have time variation due to different transmission functions, andthe time variation of the emanating light can encode information aboutthe object's spectral interactions such as the spectra in which it andother similar objects absorb, fluoresce, or otherwise interact withlight, i.e. about the type of the object.

FIG. 2 schematically illustrates general features of system 100, asystem in which light emanating from a moving object can includeinformation about characteristics of the object and in which featuresdescribed above in relation to FIG. 1 can be implemented. As with otherexemplary implementations described below, system 100 involves acombination of parts or components. Encoding component 102illustratively provides output light that includes information about oneor more object characteristics. Photosensing component 104 responds tothe output light, providing sensing results such as electrical outputsignals with information in a form that can be communicated toprocessing component 106, possibly after conversion to other forms, e.g.for storage, transmission, and processing, such as optical or otherelectromagnetic signal forms. Processing component 106 can use thesensing results from photosensing component 104 to obtain and/or providecharacteristic data indicating information about one or more objectcharacteristics.

Object 110 illustratively travels in a direction indicated by arrows112, passing through a succession of positions, two of which areillustrated. In some positions, object 110 can receive excitation,illustrated by arrows 114, and, in response, light as illustrated byarrows 116 can emanate, such as from fluorescence of a dye or other“tag” attached to object 110 or from native fluorescence orautofluorescence of object 110 itself, e.g. due to ultraviolet light orother excitation of intrinsic cell material or other material in object110; except as otherwise noted, however, implementations describedherein can additionally or alternatively employ chemofluorescence,biofluorescence, absorption, scattering, or other phenomena that do notrequire concurrent excitation. More generally, excitation could take anyappropriate form and is not limited to illumination, and excitation andemanation need not be concurrent or otherwise coincident, but could haveany appropriate relationship in space and time. Some examples ofexcitation are described below in relation to exemplary implementations.

Arrow 120 represents output light from encoding component 102. Box 122between arrows 116 and arrow 120 illustrates that information about oneor more characteristics of object 110 is included in the output light.As described below in relation to exemplary implementations, thisinformation can be encoded in a variety of ways, including, for example,patterning excitation and/or patterning emanating light to obtainencoded output light represented by arrow 120.

Arrow 120 points to photosensing component 104, indicating that at leastpart of the encoded output light is illustratively sensed by component104 to obtain sensing results. Based on the sensing results, component104 provides electrical output signals represented by arrow 130. Theelectrical output signals can also include at least some of theinformation about object characteristics from box 120. As a result,processing component 106 can, in response to the electrical outputsignals, obtain and/or provide characteristic data indicatinginformation about object characteristics.

Each of components 102, 104, and 106 in FIG. 2 could be implemented in awide variety of different ways. FIGS. 3-5 illustrate several generalfeatures of implementations of encoding component 102, each of whichinvolves an arrangement along a path traveled by a moving object.

In FIG. 3, excitation arrangement 150 is along a path traveled by movingobject 152 as it emanates light within an encoding component such ascomponent 102 in FIG. 2. As suggested by the one-dimensional coordinateaxis labeled “x OR t”, the path can be treated either as extending inspace, such as along an x-direction, or as occurring over time, t;unless otherwise indicated hereafter in relation to a specific exemplaryimplementation, the x-direction refers to an object's path and thereforemight not in some cases follow a straight line relative to theenvironment. Although the speed or other rate of displacement of object152 may vary as it travels along the path, information about its speedor other rate of displacement can be sufficient to allow an approximatemapping between its x-direction positions and times t; more generally,mapping between an object's x-direction positions and times t can bebased on any suitable system, such as with trigger detection techniquesas described in U.S. Patent Application Publication No. 2007/0145249,entitled “Sensing Photons from Objects in Channels”, incorporated hereinby reference in its entirety, or from other techniques, includingobtaining information such as a trigger signal from an object's encodedsignal.

Although excitation components could be positioned in any appropriateway along a path, the exemplary implementations described belowgenerally involve arrangements of one or more excitation componentsalong the x OR t axis, and FIG. 3 shows several exemplary componentswithin a sequence of K excitation components 154 through 156, withcomponent 154 labeled “0” and component 156 labeled “(K-1)”. Excitationcomponents need not, however, be arranged on only one side of the path,but rather could be positioned at any suitable positions around thepath, depending on how excitations from different components interact.Also, two or more excitation components could be at the same position orin overlapping position ranges along the x OR t axis, but displaced in arotation direction; a configuration of excitation components that aresufficiently displaced in a rotation direction so that they are aroundthe path is illustrated by component 158, representing a possibleposition of another excitation component labeled “(0′)” in arrangement150, on the opposite side of the path traveled by object 152 fromcomponent 154.

Arrow 160 schematically represents excitation from component 154, whilearrow 162 represents excitation from component 158. Similarly, arrow 164represents excitation from component 156. Although excitation fromcomponents 154 and 158 can be provided concurrently to object 152, assuggested by arrows 160 and 162, excitation from component 156,represented by arrow 164, is provided at a subsequent position and timeof object 152.

Excitation component 170, labeled “k1”, illustratively includes one ormore interfering light sources 171, resulting in two or more differenttypes of excitation, with two types represented by arrows 172 and 174.The excitation represented by arrow 172 occurs while object 152 travelsalong a segment of the path through region 176, while the type ofexcitation represented by arrow 174 occurs while object 152 travelsalong a subsequent segment of the path through region 178. Regions 176and 178 therefore form a pattern in space, an example of “spatiallypatterned excitation” used herein to refer to excitation that occurs ina pattern in space, i.e. a “spatial pattern”; spatially patternedexcitation could, for example, include multiple periods of a spatialpattern. In particular, the excitation in region 176 has a differentphoton energy spectrum than the excitation in region 178, so thatregions 176 and 178 could be described as having “different colors” ofexcitation. Several specific examples in which spatially patternedexcitation includes regions of different colors are described below inrelation to exemplary implementations; as will be understood from someof the examples, the x-direction of a path as shown in FIG. 3 may notfollow a straight line, so that regions 176 and 178 may not in fact beoriented along a straight line through components 154 through 156—insome implementations, regions 176 and 178 could each extend parallel tosuch a line and the path could go back and forth between regions 176 and178.

Excitation component 180, labeled “k2”, illustratively includes one ormore structured light sources 182. In other words, light sources 182 arestructured to provide spatially patterned excitation, represented byspatial pattern 186. In the illustrated example, arrow 184 representsexcitation provided in region 188, one of a pattern of regions throughwhich object 152 passes while receiving excitation from component 180.The complete pattern of regions is represented in FIG. 3 by pattern 186.

FIG. 3 also illustrates lines 190 through which each of components 154through 156 can receive control signals from excitation controlcircuitry (not shown). For example, one or more of the components inexcitation arrangement 150 could include trigger detecting circuitry(not shown) as described above, and the excitation control circuitrycould, in response to the trigger detecting circuitry, provide controlsignals causing the component to provide excitation, either in a steadystate or time-varying manner. As described below in relation toexemplary implementations, time-varying excitation can encodeinformation in a way similar to spatially patterned excitation.

Additional description of excitation techniques is set forth inco-pending U.S. patent application Ser. No. 12/023,436, entitled“Producing Time Variation in Emanating Light”, incorporated herein byreference in its entirety.

In FIG. 4, filter arrangement 200 is similarly along a path traveled bymoving object 202 as it emanates light within an encoding component suchas component 102 in FIG. 2. Filter arrangement 200 includes acombination of one or more filter assemblies along the path traveled byobject 202.

Although filter assemblies could be positioned in any appropriate wayalong a path, the exemplary implementations described below generallyinvolve arrangements of filter assemblies along the x OR t axis, andFIG. 4 shows several exemplary cross sections of filters within asequence of M filter assemblies 204 through 206, with each cross sectionbeing taken parallel to the x OR t axis and with assembly 204 labeled“0” and assembly 206 labeled “(M-1)”. Filter assemblies need not,however, be arranged on only one side of the path as shown, but rathercould be positioned at any suitable positions around the path, dependingon directional intensity variations of emanating light. Also, two ormore filter assemblies could be at the same position or in overlappingposition ranges along the x OR t axis, but displaced in a rotationdirection; a configuration of filter assemblies that are sufficientlydisplaced in a rotation direction so that they are around the path issuggested by box dashed-line box 208 in FIG. 4, representing a possibleposition of another filter assembly labeled “(0′)” in arrangement 200,on the opposite side of the path traveled by object 202 from filterassembly 204.

Filter assembly 210, labeled “m1”, illustratively includes a radialsequence of filters through which light emanating from object 202,represented by arrow 212, can pass, with the output light from filterassembly 210 being represented by arrow 214. Filter assembly 210 couldinclude any appropriate number of filters, with filters 216 and 218being shown in FIG. 4.

The overall sequence of filter assemblies 204 through 206 illustrates alongitudinal sequence. Further, filter assembly 220 includes alongitudinal sequence of filters through which light emanating fromobject 202, represented by arrows 222, can pass, with the output lightfrom filter assembly 220 being represented by arrows 224. Filterassembly 220 could include any appropriate number of filters in anyappropriate longitudinal sequence, with adjacent filters 226 and 228being shown in FIG. 4. Each of filters 226 and 228 could, for example,be a band pass filter, with the bands of filters 226 and 228 beingsufficiently different to provide useful information about an emanationspectrum of object 202. Such a filter assembly is sometimes referred toherein as a “spatially patterned filter”, because the filters itincludes can be treated collectively as a single filter that has apattern that varies as a function of position. Several examples ofspatially patterned filters are described below in relation to exemplaryimplementations, and one or both of filters 216 and 218 in assembly 210could also be implemented as a spatially patterned filter.

In the specific example of filter assembly 220, output light per arrows224 can include encoded information from filters 226 and 228, and theencoded information can be recovered by photosensing the output lightand performing appropriate operations on the sensing results. Ingeneral, filters 226 and 228 and other filters in filter assembly 220can have any suitable lengths in the x OR t direction that allowrecovery of the encoded information by photosensing and signalprocessing, including lengths smaller than the apparent extent of object202 in the x OR t direction that may result in some loss of resolutionanalogous to blurriness or smearing. As described in relation to someexemplary implementations below, however, each of filters 226 and 228can have length in the x OR t direction greater than or equal to anapparent extent of object 202 in the x OR t direction, while the lengthsof filters 226 and 228 (and other filters in assembly 220) can besufficiently small that characteristics of object 202 indicated byemanating light do not change while object 202 is traveling pastassembly 220. In some specific implementations, filters 226 and 228 haveparallel sides extending in a direction transverse to the path, and anassembly of such filters is sometimes referred to herein as a “stripedfilter” in which each stripe can be specified by filter type and itslength (or width) in the lengthwise direction.

In the specific example of filter assembly 220, output light per arrows224 can include encoded information from filters 226 and 228, and theencoded information can be recovered by photosensing the output lightand performing appropriate operations on the sensing results. Ingeneral, filters 226 and 228 and other filters in filter assembly 220can have any suitable lengths in the x OR t direction that allowrecovery of the encoded information by photosensing and signalprocessing, including lengths smaller than the apparent extent of object202 in the x OR t direction that may result in some loss of resolutionanalogous to blurriness or smearing. As described in relation to someexemplary implementations below, however, each of filters 226 and 228can have length in the x OR t direction greater than or equal to anapparent extent of object 202 in the x OR t direction, while the lengthsof filters 226 and 228 (and other filters in assembly 220) can besufficiently small that characteristics of object 202 indicated byemanating light do not change while object 202 is traveling pastassembly 220. In some specific implementations, filters 226 and 228 haveparallel sides extending in a direction transverse to the path, and anassembly of such filters is sometimes referred to herein as a “stripedfilter” in which each stripe can be specified by filter type and itslength (or width) in the lengthwise direction.

Filter arrangements similar to those shown in FIG. 4 may findapplication not only in fluidic implementations as described below butalso in implementations in which objects in an array move relative toother components due, for example, to scanning movement. One such areaof application is in image scanning, such as with scanning sheets ofpaper or other media that can bear images. In particular, object 202could be a colored spot on a sheet of paper or other medium, and afilter arrangement could be used to obtain information about smalldifferences in color of light emanating from object 202, e.g. color ofreflected light in response to broadband illumination. Such informationcould be used to obtain position and/or color of object 202; forexample, if object 202 is a registration mark with a color unique toregistration marks, its color could be accurately distinguished fromspots of other colors using techniques as described herein and itsposition could be obtained with sufficient accuracy to allowregistration of the sheet, whether for image sensing or for printing oranother operation on the sheet. Very high accuracy sensing of color issometimes referred to as “hyperspectral color sensing”.

In FIG. 5, displacement control arrangement 250 is similarly along apath traveled by moving object 252 as it emanates light within anencoding component such as component 102 in FIG. 2. Displacement controlarrangement 250 includes a combination of one or more displacementcontrol components, each of which is illustratively shown enclosing arespective segment of the path traveled by object 252. It would, ofcourse, be possible to implement display control components in otherways, such as where an object travels along a path that is not enclosedwithin a channel or fluidic structure.

Although displacement control components could be positioned in anyappropriate way along a path, the exemplary implementations describedbelow generally involve arrangements of displacement control componentsalong the x OR t axis, and FIG. 5 shows several exemplary componentswithin a sequence of control components 254 through 256, with component254 labeled “0” and component 256 labeled “(N-1)”. Although eachdisplacement control component in the sequence illustratively contains arespective segment of the path, it may be possible to implementdisplacement control components that affect displacement in overlappingsegments of a path or that interact in other ways.

Control component 260, labeled “n1”, illustratively includes shapedboundary 262, meaning that a boundary that extends partially orcompletely around the path, such as the boundary of a fluidic channel,has a shape that affects or controls displacement of object 252 as ittravels along the path, such as by affecting its speed or other rate ofdisplacement. Several examples of boundary shapes are described below inrelation to exemplary implementations.

Control component 270, labeled “n2”, illustratively includes motiondevice 272. Device 272 can illustratively cause lateral motion of aboundary in its segment of the path, as suggested by bidirectionalarrows 274. Line 276 shows that device 272 can receive control signalsfrom displacement control circuitry (not shown). Component 270 couldalso include trigger detecting circuitry (not shown), and thedisplacement control circuitry could respond to the trigger detectingcircuitry by initiating operation of device 272, either in a steadystate or time-varying manner. Examples of how device 272 could beimplemented are described below in relation to specific implementations.

FIG. 6 illustrates system 400, an exemplary system that could implementcomponents as in system 100 in FIG. 2. Although system 400illustratively includes central processing unit (CPU) 402 connected tovarious components through bus 404, a wide variety of otherarchitectures could be employed, including any appropriate combinationof hardware and software, as well as specialized hardware componentssuch as application specific integrated circuits (ASICs) for one or moreof the illustrated components or in place of a software componentexecuted by CPU 402. Furthermore, CPU 402 could be the CPU component ofany suitable machine such as a laptop or desktop computer, a specializedcomputer for system 400, and CPU 402 and other digital components asshown could be replaced by other specialized circuitry, such as ananalog signal processor; in a relatively simple application, CPU 402could be implemented with a single digital signal processor or a CPU ofa laptop or other personal computer receiving time-varying signals. Onthe other hand, in some applications, it may prove advantageous toimplement all signal processing with analog circuitry, includingoperations that compare time-varying waveforms and that obtain theirderivatives or other related waveforms, making it possible to replacesubstantially all the digital components as shown if appropriate.

System 400 also includes external input/output (I/O) component 406 andmemory 408, both connected to bus 404. External I/O 406 permits CPU 402to communicate with devices outside of system 400.

Additional components connected to bus 404 are within or connected tosystem 400. In the illustrated implementation of system 400, IC I/O 410is a component that permits CPU 402 to communicate with ICs such asphotosensing ICs; M ICs are illustrated in FIG. 6 by a series extendingfrom IC(0) 412 to IC (P-1) 414. ICs 412 through 414 illustrativelyinclude IC(p) 416 with a photosensor array 418, which includesphotosensing cells. Similarly, device 1/0 420 is a component permittingCPU 402 to communicate with various devices in system 400, such assensing and control devices; Q devices in system 400 are represented inFIG. 6 by device (0) 422 through device (Q-1) 424. In addition toexcitation components as described above in relation to FIG. 3 anddisplacement control components as described above in relation to FIG.5, devices 422 through 424 can include fluidic devices such as pumps,metering electrodes, smart gates, and other devices for gating andbifurcating, valves, flow or pressure sensors, and so forth. Suchfluidic devices could be implemented in various ways; smart gates, forexample, could be implemented with MEMS style microgates or by usingelectromagnetic forces, which are effective because most particles arecharged such that an electric field can be used to direct them asdesired in a channel.

Memory 408 illustratively includes program memory 430 althoughinstructions for execution by CPU 402 could be provided in various otherforms of software or hardware, on or off of CPU 402. The routines storedin program memory 430 illustratively include encoding routine 440;detect, readout, and combine routine 442; and object distinguishingroutine 444. In addition, program memory 430 can also store a number ofsubroutines (not shown) that CPU 402 can call in executing routines 440,442, and 444.

CPU 402 executes encoding routine 440 to encode information in lightemanating from a moving object as it travels a path, i.e. informationabout characteristics of the object. In doing so, routine 440 canprovide receive input signals from and provide output signals to devices422 through 424. For example, to obtain appropriate motion of theobject, CPU 402 can receive signals from sensors, perform computationsto determine what fluidic operations are necessary, and then providesignals to activate pumps, metering electrodes, gates, and valves toproduce appropriate relative movement between an object and othercomponents of system 400 along its path. CPU 402 can also receivesignals from trigger detecting devices and perform computations todetermine what control signals to provide to excitation components,motion devices, or other components or devices in order to performappropriate encoding in emanating light. Several examples of techniquesthat can be performed by encoding routine 400 are described below inrelation to exemplary implementations.

In executing routine 442, CPU 402 can, for example, perform pre-sensingreadout, obtain object information and sensing periods, perform sensingreadout with sensing periods and analog adjustment, digitally adjustsensing results and store quantities for an object, and combine thequantities for an object to produce its characteristic data. Routine 442could, for example, call a subroutine implemented as described in U.S.Patent Application Publication Nos. 2007/0145249, entitled “SensingPhotons from Objects in Channels”, and 2007/0146704, entitled “SensingPhotons Energies Emanating from Channels or Moving Objects”, each ofwhich is incorporated herein by reference in its entirety. Such asubroutine can be implemented for single objects moving past arrays orfor spaced multiple objects moving past arrays, provided spacingsbetween objects are sufficient to avoid interference. Also, such asubroutine can follow a general strategy of performing a series ofreadout operations, after which information for an object is combinedand its characteristic data is provided, although it would also bepossible to provide the information from each readout operationimmediately.

FIG. 7 illustrates an example of how object distinguishing routine 444in FIG. 6 could be implemented, using each object's raw data fromroutine 442 before it is used to obtain characteristic data for theobject. Routine 444 can begin with the operation in box 470, whichcoordinates routines 440 and 442 as described above, obtaining anobject's raw data, such as a data structure with photosensed quantitiesobtained from ICs 412 through 414.

The operation in box 472 receives the raw data from box 470, such as inthe form of a handle or other item of data necessary to access a datastructure. The operation in box 472 then uses the raw data to obtain theobject's characteristic data, such as in one of the ways described belowin relation to exemplary implementations. For example, an appropriatecomparison technique could be used to obtain a comparison resultindicating an object's type or other characteristic. The characteristicdata from box 472 can indicate whether the object is of interest forfurther analysis, such as because it may be suspicious or harmful or, onthe other hand, because it may be of interest for more refined analysis.

The operation in box 480 branches based on whether the object is ofinterest. If not, the operation in box 482 opens a smart gate orprovides appropriate control signals to perform another operation topurge the object from the system. But if the object is of interest, theoperation in box 484 ensures that the smart gate is closed or providescontrol signals for other suitable operations to transfer the objectdownstream so that a more refined or detailed analysis or other furtheranalysis can be performed, possibly after concentration of the objectwith other similar objects by appropriate fluidic devices.

FIG. 8 illustrates an application of a system as in FIGS. 6 and 7 inanalyzer 500 on support structure 502, a fluidic structure. Defined insupport structure 502 is serpentine channel 504 through which object 506can travel, carried by fluid such as liquid, gas, or aerosol or moved insome other appropriate way. Object 506 can, for example, be a biologicalcell or another object of any of the types mentioned above.

The manner in which object 506 enters channel 504 and is carried byfluid can be the same as described in U.S. Patent ApplicationPublication Nos. 2007/0145249, entitled “Sensing Photons from Objects inChannels”, and 2007/0146704, entitled “Sensing Photons EnergiesEmanating from Channels or Moving Objects”, each of which isincorporated herein by reference in its entirety. As explained there,object 506 can be carried through channel 504 by operation of propulsioncomponents and can be purged or otherwise caused to exit, together withfluid that is carrying it, from one of several outlets, such as throughtoggling of valves. While in channel 504, object 506 can travel througha series of sensing components, each of which can obtain informationabout object 506.

The first two sensing components after object 506 enters channel 504 areillustratively Coulter counter 510, an electrically based particle sizedetector, and Mie scatter sensor 512, also a particle size detector.Information about size of object 506 from Coulter counter 510 and Miescatter sensor 512 can be used in obtaining information about its othercharacteristics.

The next sensing component along channel 504 is emanating lightencoder/photosensor 520, shown schematically in a cross-sectional viewalong an axis similar to the x OR t axis in FIGS. 3-5, although it wouldtypically be implemented instead with components above and below channel504, similarly to other sensing components described below. Theschematic illustration of encoder/photosensor 520 includesexcitation/displacement component 522, filter component 524, andphotosensing component 526, all of which might be implemented in avariety of ways, including some of those described above and below.

After passing through encoder/photosensor 520, object 506 could becharacterized without obtaining further information, or, as in theillustrated implementation, object 506 can continue through subsequentsensing components, illustratively including components 530, 532, and534. These could, for example, include first and second fluorescencesensing components and a Raman scatter sensing component. Informationobtained from any combination of the sensing components can be used todistinguish between types of objects, such as different types ofbiological cells, or to distinguish objects from environment orbackground. Based on such a distinction, valve 540 at a bifurcationjunction can be toggled between two positions, with object 506 exitingas indicating by arrow 542 if valve 540 is in one position and exitingas indicated by arrow 544 if valve 540 is in another position.

The fluidic implementation in FIG. 8 is merely illustrative of a widevariety of implementations of the techniques described herein. Forexample, any appropriate fluidic or nonfluidic techniques could be usedwith a wide variety of different types of objects and various types ofrelative motion to gather various types of information about objectcharacteristics.

FIG. 9 illustrates an example of article 600 with components that couldbe operated similarly to encoder/photosensor 520 in FIG. 8. Somefeatures of article 600 can be understood from description in co-pendingU.S. patent application Ser. No. 11/777,712, entitled “Producing FluidicWaveguides”, incorporated herein by reference in its entirety. Forexample, article 600 includes a “fluidic structure”, used herein torefer to a structure that depends for its operation on fluid positioningor fluid flow, such as, for liquids or gases, in response to pressureor, for liquids, as a result of surface tension effects; in general, theterm “fluid” is used herein to encompass all media that can flow,including liquids, gases, aerosols, and so forth. The related term“channel” refers herein to any tube or other enclosed passage within afluidic structure through which fluid flows during operation. A channelis therefore an example of a “fluidic region”, used herein to refer to aregion that can contain fluid. An operation “positions” fluid in achannel if it changes the fluid's position in any way that leaves thefluid in the channel.

A channel or portion of a channel through which objects can travel alongpaths are treated herein as having the directional orientation describedabove in relation to a path. In addition, a “cross section” lies in aplane perpendicular to a direction in which a local net flow of fluidthrough the channel or portion can occur; a direction in which a crosssection extends can be referred to as a “transverse direction” or a“lateral direction.” A channel or portion with approximately uniformcross section and substantially linear longitudinal direction can bereferred to as “straight”, and the channels and portions describedherein are generally straight unless otherwise indicated.

In order to contain fluid, a channel or other fluidic region istypically “bounded”, meaning that surfaces or surface areas bound it onat least some sides. A “boundary” of a channel or portion is the surfaceor combination of surfaces within which fluid contained in the channelis confined. A “port” is an opening that extends through the boundary ofa channel or portion such that fluid can enter or exit through the port;in general, a port is relatively small compared to the length of thechannel or portion, and the boundary is treated as extending across theport as if the port did not exist.

As described below, article 600 can include two light-transmissivecomponents, and FIG. 9 shows article 600 in a top view through onelight-transmissive component. In this view, the inner region between thelight-transmissive components includes two main portions, channelportion 602 that can contain fluid and non-channel portion 604 thatsurrounds channel portion 602; channel portion 602 is illustrativelyshaped like a “T”, but could instead have an L-shape or any othersuitable shape, including a serpentine shape as in FIG. 8. Ports 608 areopenings through one of the light-transmissive components, allowingentry and exit of fluid into and out of channel portion 602.

FIG. 9 also shows filter assembly 610 in dashed outline. Filter assembly610 is illustratively a spatially patterned filter with a longitudinalsequence of band pass filters that includes filters 612, 614, 616, 618,and 620. Filters 612, 616, and 620 are illustratively cross-hatchedsimilarly to each other to indicate that they have the same orapproximately the same band, while filters 614 and 618 are alsocross-hatched similarly to each other, illustrating that they also havethe same or approximately the same band, a band that is different thanthat of filters 612, 616, and 620. In other words, filter assembly 610is a striped filter in which each of filters 612 through 620 can bespecified by the band that it passes and its length in the x-directionin FIG. 9.

Surrounding filter assembly 610, blocking material 622 is structured andpositioned to provide an aperture. Blocking material 622 can, forexample, be a material with approximately zero light transmission thatprevents scattering and reflection of light, also preventing lightentering filter assembly 610 from nearby fluorescing objects. Blockingmaterial 622 can be produced during the same operation that producesfilters 612 through 620 and can in effect be part of filter assembly610.

The cross section in FIG. 10 shows how light-transmissive components 630and 632 are separated by material in non-channel portion 604. Forexample, components 630 and 632 can each include quartz or anothersuitable material such as glass or acrylic with an appropriatethickness; in a successful implementation, for example, component 630has a thickness of approximately 0.3 mm, while component 632 has athickness of approximately 1.0 mm or less; depending on the application,on stability of materials used, and size of objects being characterized,suitable thicknesses might range from a few millimeters down to 0.1 mmor even less. The optimum distance between them is determined primarilyby the size of objects being characterized. For biological cells withtypical dimensions of 10 μm, for example, the distance can beapproximately 20 to 50 μm, maintained by material in non-channel portion604, which could, for example, be a suitable photoresist material suchas SU-8 or another polymer material. Alternatively, a wall (not shown)could be formed around channel portion 602, and non-channel portion 604could then be filled with epoxy material that seals a lateral boundaryaround channel portion 602. Various other techniques could be used toproduce a similar fluidic structure, including hot embossing,nano-imprinting, or injection molding, and channel portion 602 can haveappropriate dimensions, such as for waveguiding as described inco-pending U.S. patent application Ser. No. 11/777,712, entitled“Producing Fluidic Waveguides”, incorporated herein by reference in itsentirety.

FIG. 10 also shows object 640 from which light is illustrativelyemanating upward, as illustrated by an emission cone. Although theemission cone is illustratively shown as a single cone, the actualemission cone would depend on angles of total internal reflection atsurfaces through which emanating light is transmitted in article 600.FIG. 10 illustrates three alternative filter assembly positions, withfilter assembly 642 facing channel portion 602, on the lower surface ofcomponent 630; with filter assembly 644 being outside of channel 602 onthe upper surface of component 630; and with filter assembly 646 beingspaced apart from the upper surface of component 630, adjacentphotosensor 648, which could, as in other implementations, be a single,large area photosensor (such as a photo-diode, an avalanche photo-diode(APD), or a photo-multiplier tube (PMT)), or an appropriate array ofphotosensing cells whose sensed quantities can be combined to obtain asingle photosensed quantity, such as an intensity value for a sensingperiod. As suggested in FIG. 10, the emission cone from object 640 isimaged onto image plane 650 extending through filter assembly 646 byoptical component 652, illustratively shown as a single lens, but whichcould be any suitable lens, lens system, or other optical component,some examples of which are described in co-pending U.S. patentapplication Ser. No. 11/698,409, entitled “Method and SystemImplementing Spatially Modulated Excitation or Emission for ParticleCharacterization with Enhanced Sensitivity”, incorporated herein byreference in its entirety.

The emission cone for filter assembly 642 includes the range of anglesof incident light that are not totally reflected by the surface ofassembly 64. Similarly, the emission cone of filter assembly 644 isdetermined by the range of angles within which emanating light is notsubject to total internal reflection at the surface between component630 and assembly 644. The emission cone for filter assembly 646 issimilar to that for filter assembly 644, but can occupy a smaller areaon filter assembly 646 due to the effect of optical element 652.

In one illustrative example, channel portion 602 contains water with anindex of refraction n=1.33, and object 640 has a diameter d=7 μm, whichwould be typical for certain biological cells, e.g. T-lymphocytes.Channel portion 602 has a height between components 630 and 632 of 30 μmand its distance from the lower surface of filter assembly 642 isapproximately h=15 μm. Component 630 is acrylic with an index ofrefraction n=1.48, surrounded by air with an index of refraction n=1. Iffilter assembly 642 were absent, the escape angle from channel portion602 to component 630 would be a(escape)=48.75°, which would determinethe size of the emission cone in which light from object 640 can leavechannel portion 602. The angle of total internal reflection at the uppersurface of component 630, on the other hand, can be obtained asa(TIR)=42.51°, which determines the size of the emission cone for lightthat leaves component 630. The diameter of a disk illuminated by object640 at the water-acrylic interface can be obtained from D=d+2*h*tan(α(escape))=(7+(2*17.1))μm=41.2 μM, where 17.1 μm is the radius of themaximum emission cone that can pass through component 630 without totalinternal reflection. The “minimum feature size” (“MFS”) for a patternsuitable to detect object 640 at the water-acrylic interface would beequal to D or approximately 40 μm; in general, MFS can be defined for amask along the path of an emanating particle as the extent in the path'slongitudinal direction of the mask's smallest uniform feature (i.e. thesmallest transmitting filter element or the smallest blocking filterelement, whichever is smaller).

Where photosensor 648 is implemented with a numerical aperture thatmakes the emission cone smaller, filter assembly 642 can accordinglyhave a slightly smaller MFS than calculated as above; similarly, in someacrylic implementations of component 630, some light typically leavescomponent 630 at an angle slightly higher than a(TIR), which could alsoallow a slightly smaller MFS. In general, however, the MFS of filterassembly 642, if too small, results in passage of light from an object'semission cone around both sides of a feature in assembly 642, so thatthe time-varying signal of a photosensor, while containing someinformation, may not accurately indicate information about displacementof the object as it travels along a path past filter assembly 642.Similar considerations apply to filter assemblies 644 and 646, with theMFS of filter assembly 644 necessarily being significantly larger thanthat of filter assembly 642, but with the MFS of filter assembly 646possibly being intermediate between those of assemblies 642 and 644,depending on the precision of optical component 652. In implementationswithout optical components, photosensor 648 could be slightly larger dueto spreading of emanating light. For a biological cell on the order of10 μm, a typical MFS would be in the range of 10-20 μm. The channelwidth might be an order of magnitude larger, while the channel lengthmight be two orders of magnitude larger, and the width of the filterassembly would depend on the channel width. For example, assembly 642might be 100 μm wide and approximately 1.0 mm long. At the time ofmanufacture, a calibration operation could be performed using objectsthat are, for example, tiny beads with known fluorescence spectra; lightemanating from such beads could be measured and used to obtaincalibration values necessary to adjust measured values to obtain knownintensities for such objects.

The cross section in FIG. 11 further illustrates how component 630 hasoblique surface 660, a light interface surface that is illustratively atan angle of approximately 45° to the inward-facing surfaces ofcomponents 630 and 632. As a result, incident excitation light at adirection approximately perpendicular to surface 660, as illustrated byarrow 662, can cause and couple with light propagating through channelportion 602, as illustrated by arrow 664, as described, for example, inco-pending U.S. application Ser. No. 11/777,712, entitled “ProducingFluidic Waveguides”, incorporated herein by reference in its entirety.Excitation light could have any appropriate wavelength, such as 266 nm,for example. The distance from surface 660 to obtain appropriatehomogeneity can be determined, as described, for example, in U.S. PatentApplication Publication No. 2008/0013877, incorporated herein byreference; the distance can also be sufficient to allow integration ofblocking material 622.

In the illustrated implementation, the end of channel portion 602 atright in FIG. 11 is open, providing an additional port 666 through whichfluid can enter into or exit out of channel portion 602. Alternatively,article 600, instead of ending at transverse end-surface 668, couldextend to another area with ports similar to ports 608, such as with apart symmetrical about the position of surface 668; in this case, fluidcould flow through channel portion 602 between ports 608 and similarports at the opposite end of channel portion 602.

In the implementation in FIG. 11, the filters within filter assembly 610are shown in cross section, and, in this implementation, the filters donot overlap, but rather are adjacent to each other. They could, forexample, be integrated into a recess in the lower surface of component630 such that they are even with the surrounding surface of component630 or they could be surrounded on all sides by a layer of shadow (lightblocking) or transparent material of the same thickness; in either ofthese approaches, the filters could be implemented so that there is nostep at the edges of assembly 610. The size of the gap, if any, betweenadjacent filters depends, for example, on the resolution of thetechnique used to produce the filters. If the filters are produced byprinting two different light-absorbing materials that have differentabsorption spectra (in which case a surrounding layer of shadow ortransparent material could also be printed around them), theregistration and gaps between filters depend on the resolution of theprinting technique used; examples of such techniques are described inU.S. Patent Application Publication No. 2007/0172969, entitled “AdditivePrinted Mask Process and Structures Produced Thereby”, and in co-pendingU.S. patent application Ser. No. 11/755,717, entitled “Surface EnergyControl Methods for Color Filter Printing”, each of which isincorporated herein by reference in its entirety. In general, however,the techniques described herein do not require highly precisepositioning of filters—a small gap between filters should notsignificantly affect time-varying signals that result from an objecttraveling past such filters while it emanates light.

The upper part of FIG. 11 includes two graphs illustrating intensitiesdetected by photosensor 670 in response to two types of objects, oneemanating light of color “A”, the other emanating light of color “B”.Filters 612, 616, and 620 have bands that allow light of color “A” topass, while filters 614 and 618 have bands that allow light of color “B”to pass.

Curve 672 illustrates intensities indicated by sensing results fromphotosensor 670 if object 640 emanates light of color “A” as it travelsalong the path through channel portion 602. In other words, theemanating light's photon energy distribution matches the band forfilters 612, 616, and 620 so that curve 672 is high along those filtersbut low along filters 614 and 618; its high value is indicated on thevertical axis as “ImaskA”.

Curve 674, on the other hand, illustrates intensity indicated by sensingresults from photosensor 670 when object 640 emanates light of color “B”as it travels along the path. In this case, the emanating light has aphoton energy distribution that matches the band for filters 614 and 618but not for filters 612, 616, and 620, so that curve 674 is at a highintensity along filters 614 and 618, “ImaskB”, and at a low intensityelsewhere.

Curves 672 and 674 illustrate an example in which two different types ofobjects provide signals that are approximately complementary, except atthe far left along blocking material 622 where both curves are atapproximately zero intensity. In a simple implementation, for example,filters 612, 616, and 620 could be red band pass filters, filters 614and 618 could be green band pass filters, each object could either be ared fluorescing particle or tag, i.e., emanating light of color “A”, ora green fluorescing particle or tag, i.e., emanating light of color “B”.As suggested, curves 672 and 674 could be plotted based on thex-direction position of object 640 or based on the t-position within thetime varying output signal from photosensor 670, which could be providedcontinuously or by any suitable form of sampling, such as by periodicreadout at an appropriate frequency. The high intensities of curves 672and 674 would be reduced to the extent that blocking material 622prevents light from reaching photosensor 670.

As a result, output signals from photosensor 670 can be used todistinguish types of objects, in this case to distinguish objects thatemanate light of color “A” from objects that emanate light of color “B”,and examples of techniques that distinguish types of objects in variousways are mentioned below in relation to exemplary implementations. Insome examples, emanating light encoded by a filter assembly with stripesof random lengths can be analyzed by comparing a resulting time-varyingsignal with one or more templates or other signals to determine anobject's type, displacement, and position to a high level of precision.

FIG. 12 illustrates two alternative implementations similar to those inFIGS. 9-10, and with the same reference numerals, but with filterassembly 610 on a photosensitive surface of photosensor 648. Theseimplementations could be implemented by printing or otherwise depositingand patterning filters 612, 614, 616, 618, and 620 and blocking material622, such as in the manner described above, or by producing alongitudinal sequence of band pass filters in any other appropriate way,with some possible techniques being described below in relation to otherexemplary implementations. In the implementation at left, photosensor648 also operates as one side of channel portion 602, replacinglight-transmissive component 630 along at least a portion of thechannel. In other words, filter assembly 610 is positioned similarly tofilter assembly 642 in FIG. 10, allowing a very small MFS. In theimplementation at right in FIG. 12, photosensor 648 is outside ofchannel portion 602 separated from the outer surface of component 630 bya small gap of height g as shown. In this implementation, filterassembly 610 is positioned similarly to filter assembly 644 in FIG. 10,but not directly on the outer surface of component 630, so that a largerMFS is necessary. The gap between component 630 and photosensor 648 canbe maintained by spacers or other appropriate support components, andcan be sufficiently large that photosensor 648 does not interfere withanti-resonant waveguiding within channel portion 602, which can beimplemented, for example, in the ways described in co-pending U.S.patent application Ser. No. 11/316,660, entitled “Providing Light toChannels or Portions”, incorporated herein by reference in its entirety.

Absorption filters as described above in relation to FIGS. 9-12 can beimplemented in a multitude of ways. For example, rather than only twotypes of band pass filters that have bands for respective colors, threeor more types of filters with three or more respective colors could beused. Similarly, a filter assembly can include band pass filters andother types of absorption filters as would be found in a shadow mask.Furthermore, with printed filters as described above or with otherfilters produced with layers of material, overlapping band pass filterscould be produced, providing additional information. In addition,absorption filters could be combined with reflection filters, asdescribed below in relation to some exemplary implementations.

Filter assembly 700 in FIG. 13 illustrates some of these variations. Inthe illustrated assembly, each stripe is labeled with a description ofits filter criterion. Stripe 702 is a red band pass filter; stripe 704is a closed filter, meaning that it allows no transmission; stripe 706is an open filter, meaning that it allows full transmission; stripe 708is a gray filter, meaning that it passes all photon energies across arange of interest, but at an intensity in between an open filter and aclosed filter; stripe 710 is a green band pass filter; stripe 712 is acombined band pass filter that passes only the intersection of blue andgreen; and stripe 714 is a blue band pass filter. In addition, as can beseen, the widths of the stripes are random rather than periodic.

The cross section in FIG. 14 illustrates one way of implementing filterassembly 700 in FIG. 13, illustratively using patterned layers of lightabsorbing material to produce different types of filters. Theimplementation in FIG. 14 could, for example, be implemented by printingor otherwise depositing and patterning layers of material as describedabove.

In the cross section at the top of FIG. 14, filter assembly 700 includesred layer part 720, black layer part 722 overlapping layer part 720,gray layer part 724, green layer part 726, and blue layer part 728overlapping layer part 726. Where overlaps occur, the result is theintersection of two absorption filters: the intersection of layer parts720 and 722 is a closed filter, while the intersection of layer parts726 and 728 is a filter with a band that is the intersection of thebands of the green and blue filters.

The three graphs below the cross section show expected intensity signalssimilar to those in the graphs in FIG. 11. Curve 730 would be for a redfluorescing particle or tag; curve 732 would be for a green fluorescingparticle or tag; and curve 734 would be for an example where object 640is tagged both with a red and a green fluorescing particle so that curve734 is a scaled sum of curves 730 and 732. More generally, the techniqueof FIGS. 13 and 14 would make it possible to distinguish not only red,green, and blue particles and tags, but also objects tagged withcombinations such as red and green, green and blue, red and blue, andred and green and blue. Each combination results in a distinguishabletime varying signal that can be analyzed to obtain information about thecolor or colors that are emanating.

Although the intensity signals described above in relation to FIGS. 11and 14 could be obtained from sensing results of a single, large areaphotosensor, it would also be possible to use an IC with an array ofphotosensing cells or an array of discrete photosensors, in either caseappropriately positioned along a path traveled by objects past one ormore filter assemblies. If an array is used, and each element of thearray is covered with a different filter assembly, it may be possible todistinguish many different types of particles concurrently. The numberof particles to be distinguished can be much larger than the number ofelements in the array, since each measurable distinguishing feature canprovide one axis in a principal component analysis, and multipleparticles can be distinguished along each such axis. Additionaltechniques that can be used to track and distinguish objects aredescribed in co-pending U.S. patent application Ser. No. 11/702,328,entitled “Distinguishing Objects”, incorporated herein by reference inits entirety. Objects can be distinguished, for example, from theirenvironment or background or from objects of other types; an operation“distinguishes” objects if the operation locates, selects, sorts,counts, or otherwise identifies an object or controls or directs anobject according to type or separates objects or otherwise treatsobjects differently in some way.

Band pass filters of other types can also be used to implement filterassemblies as described in some of the exemplary implementations herein.For example, interference based filters can have different bands similarto the bands described above in relation to FIGS. 9-14.

Filter assembly 750 in FIG. 15 illustrates an implementation in which athin layer of transparent material creates Fabry-Perot oscillations, andcan be structured to obtain high thickness-dependent index contrast.Assembly 750 includes filters 752, 754, 756, 758, and 760, each of whichhas substantially constant thickness, but with the thicknesses offilters 752, 756, and 760 being approximately equal to each other whilethe thicknesses of filters 754 and 758 are approximately equal to eachother but smaller. Assembly 750 could be produced, for example, byetching a deposited layer of transparent material or by imprinting anon-solid layer of such material before it solidifies.

To the right of the cross section of assembly 750 is a graph showing anintensity-energy function of its transmitted light. In other words,curves 770, 772, and 774 are approximately the same because filters 752,756, and 760 have approximately the same thickness. On the other hand,curves 774 and 776 are also similar to each other but different than theothers, because the thicknesses of filters 754 and 758 are the same aseach other but different than the others. As a result, an objecttraveling along a path past assembly 750 results in a time-varyingsignal with changing intensity-energy function. The total transmissionat each position will relate to the overlap of the cavity's transmissionlines and the particle spectrum. The other part of the emanating lightwould be reflected from assembly 750, and could also be detected toobtain confirming information. For example, assembly 750 could be on onecover slide of a channel, and two photosensors (not shown) could bepositioned, one on the side of assembly 750 away from the channel andthe other on the opposite side of the channel to obtain sensing resultsfor the reflected emanating light.

FIGS. 16 and 17 illustrate two ways in which Fabry-Perotinterference-based filters could be structured to obtain band passfilters more nearly similar to those of FIGS. 9-12. In eachimplementation, optical thickness of the filter's cavity varies in thex-direction, but the variation in optical thickness is produced in twodifferent ways. The general strategy in FIGS. 16 and 17 is to provideregions that operate as band pass Fabry-Perot filters, with differentsets of filters having transmission peaks at different photon energies.For example, one set of filters could have a transmission peak atapproximately 822 nm, while another could have a transmission peak atapproximately 833 nm, and the two sets could have a periodic pattern asshown or any other appropriate pattern.

Filter assembly 800 in FIG. 16 includes homogeneous bottom distributedBragg mirror (DBR) 802, cavity 804, and upper DBR 806. Such a filterassembly could be produced by using techniques described in U.S. Pat.No. 7,315,667, entitled “Propagating Light to be Sensed”, incorporatedherein by reference in its entirety. As can be see in FIG. 16, however,the optical thickness of cavity 804 has been modified by changingbetween two thicknesses, one larger and one smaller, so that assembly800 effectively includes two sets of filters: reference numerals 810,812, and 814 indicate three filters with the larger thickness whileregions 816 and 818 are filters with the smaller thickness, thereforetransmitting a shorter wavelength than the filters in regions 810, 812,and 814. The variations in thickness of cavity 804 can be produced, forexample, by etching the layer in which cavity 804 is formed after it isdeposited and before the series of layers in DBR 806 are deposited.Alternatively, a half-tone mask could be used during growth of cavity804.

Filter assembly 820 in FIG. 17 similarly includes lower DBR 822, cavity824, and upper DBR 826, each of which illustratively has approximatelyuniform thickness, but with cavity 824 having optical thickness thatvaries in the x-direction. As a result, regions 830, 832, and 834transmit a different photon energy than regions 836 and 838. Morespecifically, the refractive index of cavity regions 840, 842, and 844is different than the refractive index of regions 846 and 848.Differences in refractive index could be produced in a wide variety ofways. Implantation or ion diffusion (as in ion exchange) could beperformed as is done in fabricating waveguides for integrated optics;another approach would be implantation-induced intermixing of multiplequantum well (MQW) structures as in laser diode fabrication; further,ultraviolet light-induced changes in refractive index could be used aswith germanium-doped glass used in fabricating fiber Bragg gratings(FBG) in glass fibers; in principle, any technique that can modifyrefractive index by implantation, heat, light, or other operation couldbe used.

FIG. 18 illustrates an additional technique that could be used togetherwith the technique of FIG. 15 and possibly the techniques of FIGS. 16and 17. Filter component 860 includes a wedge-shaped layer oftransparent material as in FIG. 15 or Fabry-Perot filter as in FIGS. 16and 17, but with filter assemblies 864, 866, and 868 formed at its uppersurface such as by techniques described in relation to FIG. 15, 16, or17. In other words, in addition to having filters of the types describedabove, there is also a continuously varying thickness across component860 so that, in addition to the time-varying effects of each filterassembly, additional spectral information is contained in the encodedemanating light, and can be obtained by appropriate processing. Withtechniques such as this, it may be possible to measure the entirespectrum with a loss of not more than 50% (assuming full modulation) ofthe light, which would be advantageous in comparison with conventionallinear variable filter approaches.

In implementations as in FIGS. 9-12, laminar flow can be used to providesubstantially uniform object speed past a filter arrangement. Incontrast, FIGS. 19-21 illustrate examples in which laminar flow canproduce non-uniform displacement or can be modified in other ways.

FIG. 19, taken along a line similar to line 19-19 in FIG. 12, showswall-like parts 870 and 872 with linearly decreasing distance betweenthem. As a result, as object 640 passes along two-color filter assembly610 (with filter elements 612, 614, 616, 618, and 620 illustrativelyperiodic rather than random as in FIG. 12), its velocity increaseslinearly as indicated by curve 874, either as a function of position orof time. Therefore, rather than a periodic time-varying signal, theresulting time-varying signal is chirped, meaning that the periodsdecrease linearly due to change in velocity of object 640 due to changein the flow speed of fluid in the channel resulting from the changingchannel dimensions. Curve 874 illustrates the resulting chirped signal,which has intensity I(A) during regions 612, 616, and 620, and intensityI(B) during regions 614 and 618. As can be seen, the duration of thesignal during each successive region is shorter than the precedingregion, resulting in the chirped pattern. For the sake of illustration,the linear decrease in transition time is exaggerated in curve 874 incomparison to the narrowing of the channel.

The technique in FIG. 19 is only one of a variety of ways of producing achirped time-varying signal, and various other techniques could be used.For example, more complex flow speed distributions could be obtained bymodifying the channel walls in other ways or by providing devices thatchange the flow speed or flow pattern within the channel, any of whichwould produce more complex time-varying signals from different objects.

FIG. 20 illustrates, on the other hand, how relatively simpletime-varying signals could be produced using more complicatedtechniques. In general, such techniques assume that geometry of achannel directs flow of object 640 in a defined manner such as periodic,chirped, or random, past a sequence of filter elements. This allowsredirection of particle flow past a simpler filter assembly geometry,and may be advantageous in cases where it is easier to redirect particleflow to produce a desired time-variation of emanating light than itwould be to produce a filter assembly to produce the same timevariation; for example, it might be easier to change channel wall shapesthan to produce a desired filter assembly. In other cases, on the otherhand, it might be advantageous to obtain more abrupt or rapid signaltransitions with a well-defined filter assembly. In addition to thetechniques described below, which involve shaping or moving walls, anobject's flow within a channel could also be redirected by othertechniques; an electrically charged object such as a particle, forexample, could be redirected by electrical field variations. In general,however, the Reynolds number in typical microfluidic and nanofluidicimplementations are so small that laminar flow conditions are, as apractical matter, always present.

In the example in FIG. 20, wall-like parts 880 and 882 are parallel buteach of them is shaped like a sinusoidal wave, resulting in a sinusoidalflow pattern in the channel between them. Filter elements 884 and 886are homogeneous of two different colors, illustratively labeled “A” and“B”. As object 640 follows sinusoidal path 888, it moves back and forthbetween elements 884 and 886, passing through a small gap between themtwice during each period. Curves 890 and 892 illustrate exemplarytime-varying signals that could result from an object traveling alongpath 888. Curve 890 illustrates an example of an object of a type with aspectrum similar to color A but different from color B, while curve 862illustrates an example of an object of a type with a spectrum similar tocolor B and different from color A. As a result, the curves are somewhatcomplementary, although each curve goes to approximately 0 while path888 is crossing stripe 894 of blocking material between elements 884 and886. Blocking material could also be provided outside elements 884 and886.

Wall-like parts 900 and 902 in FIG. 21 are substantially straight andparallel, with filter elements 904 and 906 between them, similar toelements 884 and 886 in FIG. 21. Motion device 908 produces relativemovement between the path of object 640 and stripe-like elements 904 and906, as indicated by bi-directional arrow 910. Control circuitry 912provides signals to control operation of motion device 908, which neednot be periodic, but could take any appropriate pattern, resulting inarbitrary time-varying signals with features indicating different typesof objects. An alternative would be to move elements 904 and 906; moregenerally, any combination of relative movements between walls 900 and902 on the one hand and elements 904 and 906 on the other could producemovement as indicated by bi-directional arrow 910. Furthermore,additional variations could be produced by changing fluid flow withinthe channel so that the speed or other displacement of object 640changes as a function of time relative to the other movements. Motiondevice 908 could be set up to produce variations in response to triggersignals indicating incoming objects.

Curve 914 illustrates movement of object 640 between element 904,labeled “Band A”, and element 906, labeled “Band B”. As illustrated,object 640 spends different lengths of time in each region and can spenda random amount of time in each region, resulting in a random excitationpattern. Curves 916 and 918 illustrate exemplary time-varying signalsthat could be produced by the technique of FIG. 21. One type of objecthas a spectrum more similar to color A of element 904, as illustrated bycurve 916, while the other has a spectrum more similar to color B ofelement 906, as illustrated by curve 918. As each object travels betweenelements 904 and 906, it passes over stripe 919 of blocking materialbetween them, resulting in a brief interruption of the emanating light,so that each curve goes briefly to 0. In curve 916, the intensity alongelement 904 is I(A1), while the intensity along element 906 is I(B1), alower value linearly. Conversely, curve 918 illustrates that theintensity is higher along element 906, at intensity I(B2), and loweralong element 904, at intensity I(A2). The two curves are, in general,complementary, except for times when they are passing stripe 919 betweenelement 904 and 906; object 640 can be moved instantaneously betweenBand A and Band B, moving very quickly across stripe 919, so that thetime in which it is passing stripe 919 are very brief.

FIGS. 22-24 illustrate implementations of filter arrangements in whichfilter assemblies are on opposite sides of channel 920. In theillustrated implementation, detector 922, shown on the near side ofchannel 920, includes one filter assembly, while detector 924, on thefar side of channel 920, includes another filter assembly. Although eachdetector could be implemented in a wide variety of different ways, toobtain information about emanating light and objects from which lightemanates, FIGS. 23 and 24 illustrate an example in which detector 922includes a periodic filter assembly with periodicity in a directiontransverse to channel 920, labeled the y-direction, and detector 924includes a random two-color filter assembly with a longitudinal sequencein the x-direction, though other angles between the x- and y-directionsmight also be useful including, in some cases, implementations in whichthey are parallel. In the illustrated case, sensing results fromdetector 922 include signals modulated in the y-direction, while sensingresults from detector 924 indicate signals modulated in the x-direction.The two modulations can be used to obtain information about an objectfrom which light is emanating.

As shown in FIG. 23, detector 922 can be implemented with photosensor926 on a photosensitive surface of which are filters 927, periodic inthe y-direction; each of filters 927 is illustratively a red band passfilter, but they could instead be any other color or closed filters orintermediate intensity gray scale filters, and could be implemented withabsorption, reflection, or interference-based filtering techniques asdescribed above. Similarly, FIG. 24 shows an implementation of detector924 in which photosensor 928 has filter assembly 610 (FIG. 9) on itsphotosensitive surface; photosensor 928 could also have a periodicfilter superimposed on filter assembly 624 or in place of filterassembly 624, in which case it might include green filters (not shown).

A wide variety of other arrangements similar to FIGS. 22-24 would bepossible, including, for example, another type of template layer on oneside of channel 920 to provide a desired signal as described inco-pending U.S. patent application Ser. No. 12/022,485 entitled“Obtaining Information from Time Variation of Sensing Results”,incorporated herein by reference in its entirety, and a periodic masklayer to provide a periodic signal on the other side of channel 920; inthis implementation, the periodic signal could be constantly analyzed toobtain values indicating displacement of an object currently flowingthrough channel 920, which could be used to determine an appropriatetime scale for correlation with the template signal similar totechniques described. In another possible variation, emanating lightfrom fluorescence could be photosensed on one side of channel 920 andemanating light due to scattering, for example, could be photosensed onthe other side.

Some of the exemplary implementations described below involve filterassemblies that combine periodic signals additively with templatesignals from filter sequences similar to some of those described above.The resulting time-varying signal emerges from the filter assembly withtwo different spatially varying patterns imposed on it. To produce sucha signal, for example, a radial sequence or “stack” of filters similarto that shown in FIG. 4 could be used. Within a stack of filters, forexample, one layer could be a template layer with an appropriate patternto produce the template signal, while another layer could be a periodiclayer with an appropriate pattern to produce the periodic signal; eachof the template layer and periodic layer could have rectangles or otherclosed polygons of zero opacity surrounded by regions with opacity 0.5.

FIGS. 25 and 26 illustrate an alternative approach that can be used withreflective gray scale filters, producing a single filter assemblyequivalent to a desired radial sequence or stack of filters. To obtainfilters as in FIGS. 25 and 26, thickness definitions of two filterlayers can be overlaid using software tools and the thicknesses ofoverlapping regions can be added, resulting in regions with thicknessesof 0, 0.5, and 1 in the example given above; the two filter layers couldboth be oriented with variation in the same direction as in FIGS. 25 and26, similar to the techniques of FIGS. 9 and 13, or could be orientedwith variation in different directions, e.g. orthogonal to each other.For implementations in which layer thickness does not appropriatelydefine or determine the desired equivalent filter's structure or itsoptical variation, the techniques in FIGS. 25 and 26 could be modifiedto first overlay optical feature definitions of the filters in whichregions have defined optical feature values that determine the desiredvariation, thus obtaining an optical feature definition of the desiredequivalent filter; the optical feature definition could then beconverted to a layout-type description of the equivalent filter in whicheach region has a defined optical thickness or other characteristic thatcan be produced to provide the region's value for the optical feature.

The techniques of FIG. 25-26 take advantage of the fact that, ingeneral, superpositions of filters are commutative, in the sense thatthe resulting transmission or reflection function is the same regardlessof the order in which filters are superimposed. There are, of course,exceptions, such as where interference effects can occur if filters arein a specific order, or where alignment or other relationship of filterfeatures can result in loss of different information depending on theorder of the filters.

If the equivalent filter definition is a thickness definition to producea purely transmissive/reflective filter with no color variation, and ifpartial etching can be performed, an equivalent filter that approximatesthe equivalent filter definition can be constructed by first depositinga highly reflective material, such as chromium, over the entire filterassembly, and by then partially etching the reflective material away inregions with thickness 0 or 0.5 to an appropriate extent, leaving athin, partially transmitting layer, after which the remaining reflectivematerial can be etched away in regions with thickness of 0. Wherepartial etching is unreliable, other techniques may be used, such as bytechniques that deposit a first patterned layer of thickness 0.5 withany suitable patterning technique, then depositing over it a secondpatterned layer of thickness 0.5 that is patterned without etching, suchas with liftoff or other patterning techniques that do not requireetching. Furthermore, similar techniques might be applied to producelayered filter structures that include DBRs of varyingtransmission/reflectivity and/or cavities of varying optical thickness,such as those described above in relation to FIGS. 16-18; variation incavity thickness could result from any appropriate combination ofthickness variation and refractive index variation, produced with anyappropriate techniques.

Filter 930 in FIG. 25 is equivalent to the combination of a randomfilter and a periodic filter, superimposed one on the other. Curve 932shows the shape of the random filter, while curve 934 shows the shape ofthe periodic filter; as can be seen, the random and periodic filtersboth have only two thickness levels, either 0 or 0.5, but filterassembly 930 has three thickness levels, corresponding to 0, 0.5, and 1.Curve 936 shows a resulting transmission function. Emanating lightpassing through filter assembly 930 includes both displacement andposition information about an object from which it emanates, and allowstime-scaling techniques to extract that information, as described below.

The technique illustrated in FIG. 25 can be adjusted as suggested bydashed lines 938 within filter 930. In other words, total light outputcan be changed by scaling the amplitude of the thickness levels: ratherthan 0, 0.5, and 1, for example, thickness levels of 0, 0.2, and 0.4could be used, allowing greater light transmission. It may be necessary,however, to make a tradeoff between greater light output, and thereforetotal signal intensity, on the one hand, and greater light modulation onthe other—greater light modulation may facilitate calculation ofdisplacement and position within a given observation region. The masksuggested by dashed lines 938 emphasizes total light output because ithas reduced thickness and, conversely, increased transmission, with athickness of 0 being equivalent to transmission of 1 and vice versa. Thescaling suggested by dashed lines 938 may require great precision: thex-direction scale of features in assembly 900 may be as great as 10 μm,while a useful thickness may be as thin as 10 nm of chromium.

Similarly, filter assembly 940 in FIG. 26 is equivalent to thecombination of a chirp filter represented by curve 942 and a periodicfilter represented by curve 944. A combination of chirp and periodicfilters can make it possible to more efficiently extract displacementand position information about objects that may have different speeds.Curve 946 shows a resulting transmission function, which allowsinformation extraction.

A stack-equivalent filter assembly as in FIGS. 25 and 26 can in somecases have a smaller MFS than either of the simpler non-uniform filters.As mentioned above, loss of resolution can occur for light emanatingfrom objects approximately as large as the MFS.

FIG. 27 illustrates one way in which a longitudinal sequence of filters,such as a random band pass filter arrangement as described above inrelation to FIGS. 9-12 can be combined with a reflective gray scalefilter arrangement, illustratively a periodic gray scale filter. Filterarrangement 950 in FIG. 27 includes filter subassembly 952 with alongitudinal sequence similar to that described above in relation toFIGS. 9-12. On the upper surface of subassembly 952 is a periodic filtersubassembly with regions 954, each having an intermediate transmissionlevel such as 0.5. As a result, filter assembly 950 combines thetechnique of FIG. 25 with that of FIG. 12, providing distinguishabletime-varying signals for emanating light of different colors, and alsomodulating the emanating light to allow time-scaling techniques asdescribed below. In effect, the time-scaling operations can be performedin the same way for each emanating color's signal, and the differentcolor signals can be used to distinguish types of objects after timescaling.

The flow chart in FIG. 28 illustrates ways in which information aboutobjects can be obtained and used by CPU 402 (FIG. 6); the technique ofFIG. 28 illustratively extracts information such as a type, a position,or a spectral difference, and uses such information to distinguishobjects. FIG. 28 also suggests ways in which routines 440, 442, and 444(FIGS. 6 and 7) could be implemented. Although suitable for CPU 402,operations in FIG. 28 could be implemented with a wide variety ofdifferent types of circuitry with or without a CPU. Furthermore,although described in terms of time-varying signals from photosensors,the technique of FIG. 28 could be applied to any time-varying sensedsignals, including, for example, capacitively sensed signals fromcharged particles with encoded information due to shapes, sizes, andpositions of electrodes.

The operation in box 970 obtains one or more encoded time-varyingsignals from a photosensor arrangement as one or more objects travelalong respective paths past a filter arrangement. The technique could beimplemented with a single photosensor along the paths, but it might alsobe possible to implement with two photosensors on opposite sides of thepaths or with other photosensor arrangements. The objects can, forexample, travel through a channel as described above in relation toFIGS. 8-12 and 19-21 and the time-varying signals can be encoded in anyof a wide variety of ways using filter arrangements, including one ormore of those described above, with or without displacement controland/or spatially modulated excitation; excitation techniques that couldbe used are described in co-pending U.S. patent application Ser. No.12/023,436 entitled “Producing Time Variation in Emanating Light”, alsoincorporated herein by reference in its entirety. For example, if one ofthe filter arrangements is at least partially non-periodic or ifdisplacement control or excitation is at least partially non-periodic, arespective template of the resulting non-periodic pattern for each of anumber of types of objects can be used to perform a correlationoperation; in other implementations, two differently encodedtime-varying signals can be obtained in box 970 and correlated with eachother. Note, however, that two types could be distinguished based on asingle template, especially if their time-varying signals aresufficiently complementary that one results in correlation and the otherin anti-correlation with the template.

The operation in box 970 can include providing any appropriate controlsignals to other components of the system, including signals to read outsensing results of photosensors. The control signals could beimplemented as in routines 440 and 442 (FIG. 6), with CPU 402 providingsignals through device I/O 420 to one or more of devices 422 through424. For example, fluid flow speed could be adjusted and channel wallmovement could be controlled as described above in relation to FIG. 21.In order to obtain the time-varying signals, CPU 402 could providesignals through IC I/O 410 to obtain photosensed quantities from ICs 412through 414.

The operation in box 972 performs a correlation or other comparingoperation on one or more time-varying signals from box 970, such ascomparing two encoded signals with each other or comparing one encodedsignal with a respective template of a non-periodic encoding pattern foreach distinguishable type of object. As used herein, the term“correlation operation” encompasses any of a variety of mathematicaloperations that can be performed on a pair of time-varying functions,with or without scaling, and that obtains a similarity measure as afunction of time-alignment. This correlation operation can beimplemented, for example, as described in co-pending U.S. patentapplication Ser. No. 11/698,338, entitled “Method and System forEvaluation of Signals Received from Spatially Modulated Excitation andEmission to Accurately Determine Particle Positions and Distances”,incorporated herein by reference in its entirety. Additional correlationand other comparison techniques that could be used are described inco-pending U.S. patent application Ser. No. 12/022,485 entitled“Obtaining Information from Time Variation of Sensing Results”, alsoincorporated herein by reference in its entirety.

A correlation operation in box 972 can produce correlation results foreach pair of waveforms that is compared. For example, if box 972compares an encoded time-varying signal from box 970 with each of Ntemplates for N types of objects, N correlation results are produced.

The graphed curves in box 974 illustrate two types of correlationresults: The upper curve illustrates a correlation result where twotime-varying waveforms are correlated, i.e. highly similar at the timealignment designated t_(p); the lower curve illustrates a correlationresult where two time-varying waveforms are anti-correlated, i.e. highlydissimilar at the time alignment designated t_(p)′. In each case thereis a peak, with the peak in the correlated case marked to show itsamplitude A and with the anti-correlated case having an inverted peak ofsimilar amplitude. If correlation is performed on a continuous basis,correlation results could similarly be continuously obtained for eachtemplate with which comparison is made, with each object's travel pastthe filter arrangement producing a peak, an inverted peak, or a featurein between the two for each template.

The operation in box 980 obtains a time-varying waveform that equals orapproximates the time derivative d/dt of each correlation result frombox 972. For the correlated case, a derivative waveform like the graphedcurve in box 982 is obtained, with a positive peak followed by anegative peak, with a zero crossing at t_(p), and with the contrast ordifferential quantity between the peaks again being the amplitude A. Forthe anti-correlated case, a derivative waveform like the graphed curvein box 984 is obtained, with a negative peak followed by a positivepeak, with a zero crossing at t_(p)′, and with the contrast ordifferential quantity between the peaks being amplitude A′, theamplitude of the inverted peak in the lower graph in box 974. Theamplitudes obtained in this manner are, in general, free of offsets,allowing direct comparison to obtain spectral information.

The operation in box 986 uses derivative waveforms from box 980 toextract information for objects passing the photosensor. The extractedinformation could, for example, be a type based on whether an objectresulted in correlation, anti-correlation, or neither with a giventemplate; position based on the time at which a zero crossing occurs incorrelation or anti-correlation; and spectral difference, e.g. adifference of emission, absorption, or scattering spectrum, based on theamplitude or contrast between positive and negative peaks fromcorrelation and anti-correlation, respectively. Features of a derivativewaveform could be found and measured using various techniques. Theoperation in box 988 can then be performed to distinguish objects usinginformation extracted in box 986, such as by obtaining counts ofdifferent types of objects or ratios between such counts, or with otheroperations as described above in relation to FIG. 7.

The operations in boxes 972, 980, and 986 could be implemented, forexample, as parts of one or both of routines 442 and 444 (FIG. 6). Theoperation in box 988 could be implemented as part of routine 444. Ingeneral, these operations could be implemented to handle signals fromeach object separately or to handle a signal received concurrently or inseries from a number of objects, in which case minimum differences, suchas in positions or speeds, may be necessary to allow separation ofsignals from different objects. Any appropriate combination of serialand parallel operations could be implemented in any appropriatecircuitry. Data streams or other data structures defining waveforms suchas templates could be stored and retrieved as needed by routines 442 and444, such as in memory 408 (FIG. 6). Similarly, intermediate and finalresults of operations in boxes 972, 980, 986, and 988 could similarly bestored and retrieved as needed.

Comparison techniques other than correlation could be employed, butcorrelation techniques can be advantageous because they are typicallynot sensitive to noise, such as an AC power frequency. For example,preliminary smoothing or other preprocessing of waveforms is typicallyunnecessary for correlation, and available techniques for computingcorrelations can produce useful results even with S/N ratiossignificantly less than 1.0. It is, however, necessary to satisfyminimum sampling requirements if waveforms are digitized forcorrelation; in accordance with the Nyquist frequency, each waveformshould be sampled at least twice during the time duration of its minimumfeature size.

Some techniques as described above have been successfully applied tosimulated time-varying waveforms. In particular, time scaling techniqueshave been found to improve S/N ratio of a simulated observed signal thatcontains both an encoding based on a template and also additive noise,and where the observed signal has an unknown time scaling that occursbefore it is observed; S/N ratio of 0.5 has been obtained and 0.1appears achievable. These results could be obtained with particle speedsup to 0.5 m/sec and higher speeds up to a few msec appear to befeasible, with particles having effective sizes down to 0.6 μm, and withparticle separations down to a given implementation's MFS. Ademonstration included counting CD4 in a whole blood sample; single tagdetection was shown to be feasible.

Where a simulated observed signal includes or is accompanied by asimulated concurrent periodically modulated signal, time scaling of atemplate waveform based on a scaling factor from the periodicallymodulated signal has successfully produced matching correlation results,indicating correlation or anti-correlation as appropriate and makingspectral information available, in effect allowing concurrent detectionof multiple colors with a single detector such as a large-areaphotosensor. Because an object receives different excitations at almostthe same time and location (due, for example, to interdigitated orotherwise patchworked or patterned excitations), differences inabsorption and excitation spectra can be measured with very highprecision; similarly, because different spectral subranges of anobject's emission spectra are measured at almost the same time andlocation (due, for example, to interdigitated, or otherwise patchworkedor patterned filter arrangements), differences in emission spectra canbe measured with very high precision; therefore, with one or both ofpatterned excitation and patterned filtering, many types of errorscancel out, including time-dependent factors such as bleaching,intermixing, diffusion and also errors induced by excitation differencessuch as temperature gradients and optical misalignments. Particleposition can be precisely determined from fine structure of correlationresults. As noted above, simulation results show that spatial resolutionof less than 1.0 μm is possible, and single fluorescence markers can bedetected, making detection possible with smaller amounts of consumablessuch as markers. The techniques appear appropriate for nativefluorescence, allowing agent-less detection.

Some of the implementations described above in relation to FIGS. 1-28are examples of a method of using a filter arrangement. While an objecttravels along a path past the filter arrangement and emanates lightwithin an application's range of photon energies, the methodtransmits/reflects at least some of the emanating light through thefilter arrangement. In doing so, the method includes at least one of thefollowing two: First, while the object is in each of two or moresegments of the path, the method transmits/reflects respective portionsof the emanating light through respective positions of a filter assemblywithin the filter arrangement; each of the respective positions has arespective transmission function, and the transmission functions of atleast two of the positions are sufficiently different that timevariation occurs in the emanating light between at least two of thesegments. Second, while the object is in each of a series of segments ofthe path, the method transmits/reflects a respective portion of theemanating light through a filter component within the filterarrangement, and the filter component has a combined transmissionfunction in which a set of simpler transmission functions issuperimposed; the set of transmission functions is superimposed suchthat time variation occurs in the emanating light in accordance withsuperposition of first and second simpler non-uniform transmissionfunctions in the set.

In specific implementations, the method can do both, i.e. ittransmits/reflects respective portions of the emanating light throughrespective positions of a filter assembly and also transmits/reflects arespective portion of the emanating light through a filter component.The filter assembly can include first and second filter elements thatinclude first and second respective positions, and each of the first andsecond filter elements can have a respective transmission function thatis approximately uniform for light from its respective segment of thepath. For example, the transmission functions of the first and secondfilter elements can be spectrally different, and the method can encodespectral information in time variation of the emanating light. Also, thetransmission functions can be different in transmitted/reflectedintensity, and the method can encode intensity information in timevariation of the emanating light. More generally, the method can encodeinformation about the object in time variation of the emanating light,such as information indicating the type of the object.

In further specific implementations, each of the segments of the pathcan be at least approximately as large as the object's size. Thecombined transmission function can have a minimum feature size (MFS) atleast approximately as large as the object's sizes. The series ofsegments in which the method transmits/reflects emanating light throughthe filter component can be substantially continuous. Similarly, thesegments from which emanating light is transmitted/reflected throughrespective positions of the filter assembly can be segments within asequence in part of the object's path. The method can cause the objectto travel along the path with non-uniform displacement, such as bychanging its displacement rate or its displacement direction. The objectcan be, for example, a biological cell or virus, and the application canbe flow cytometry.

The simpler non-uniform transmission functions that are superimposed caninclude at least one that is non-periodic. Examples of simplertransmission functions can include, in addition to periodic functions,random and chirped functions.

Some of the implementations described above in relation to FIGS. 1-28are examples of apparatus that includes a fluidic structure with achannel through which objects can travel along respective paths duringoperation and an encoding component with a filter arrangement that canreceive light emanating from objects in the channel. In response toinput light emanating from an object, the filter arrangement providesoutput light. The filter arrangement includes at least one of thefollowing two: First, a filter assembly with a set of positions, eachhaving a respective transmission function; a sequence of segments of anobject's path including at least two segments from which respectivepositions in the set receive emanating light, and the transmissionfunctions of the positions are sufficiently different from each otherthat time variation occurs in the output light while the object travelsthrough the sequence of segments. Second, a filter component thatreceives input light from a segment of an object's path and has acombined transmission function in which a set of two or more simplertransmission functions are superimposed; the set includes first andsecond simpler non-uniform transmission functions, and the set issuperimposed such that time variation occurs in the output light inaccordance with superposition of the first and second simplernon-uniform transmission functions while the object travels through thesegment.

In specific implementations, the apparatus can encode information aboutthe object in time variation of the output light. The apparatus can alsoinclude a photosensing component that photosenses the time-varyingoutput light and provides sensing results, such as with electricalsignals. The apparatus can also include a processing component thatresponds to the sensing results, performing operations to obtain dataindicating information encoded in the output light. The processingcomponent can be programmed, for example, to perform a comparingoperation to obtain comparison results between time-varying waveforms,at least one of which is from the sensing results, and can use thecomparison results to obtain data indicating at least one spectraldifference between the time-varying waveforms; the comparing operationcan be correlation, and the time-varying waveforms can include a sensedtime-varying waveform and a template time-varying waveform.

In specific implementations, the filter arrangement can include one ormore of many different filter components, such as an absorption filter;an interference-based filter; a light-transmissive and/orlight-reflective filter; a longitudinal sequence of filters that vary ina periodic, random, or chirped pattern; filter elements of two or morecolors; filter elements of two or more gray levels; overlapping filterelements; a longitudinal sequence of filter elements that includesbinary, gray level, and color filter elements; two or more lengthwiseextending filter elements; a radial sequence of filter elements; twoorthogonally striped filter assemblies; filter assemblies on oppositesides of a fluidic channel; and a stack-equivalent filter. The filtercomponent can include the filter assembly, which can have one of thefirst and second simpler non-uniform transmission functions. At leastone of the simpler non-uniform transmission functions can be a periodic,random, or chirp function of position.

In specific implementations, the apparatus can be a flow cytometer, withthe objects being, for example, biological cells or viruses. In otherimplementations, the apparatus can be a scanning device.

Some of the implementations described above in relation to FIGS. 1-28are examples of a method that transmits/reflects light emanating fromobjects through a filter arrangement while each object travels along arespective path past the filter arrangement. The method cantransmit/reflect at least some of the object's emanating light through alongitudinal sequence of filter elements within the filter arrangement,while the object travels through a segment of its path. The longitudinalsequence includes a first subset of filter elements that haveapproximately a first transmission function and a second subset thathave approximately a second function, with the first and secondtransmission functions being sufficiently spectrally different from eachother that time variation occurs in the emanating light while the objecttravels through the segment.

In specific implementations, the time variation encodes spectralinformation about the object, and the method can photosense theemanating light to obtain photosensing results, then use thephotosensing results to obtain data indicating the encoded spectralinformation. The photosensing results can indicate sensed time-varyingwaveforms, and the method can perform a comparing operation on a set oftime-varying waveforms to obtain comparison results, with at least oneof the time-varying waveforms being a sensed time-varying waveform; themethod can use the comparison results to obtain data indicating at leastone spectral difference between the time-varying waveforms.

In further specific implementations, where objects have differentrespective emanation spectra, the method can detect a difference betweenthe spectra. The longitudinal sequence can be a sequence of spatiallypatterned filter elements that includes at least one spatially patternedcolor filter element and at least one spatially patterned non-colorfilter element such as a black and white or gray level filter element.More generally, a sequence of patterned filter elements can includepatterns sufficiently different that different information items areconcurrently encoded in the emanating light without loss of information.

Some of the implementations described above in relation to FIGS. 1-28are examples of a method that transmits-reflects light from objectsthrough a filter arrangement while the objects travel past the filterarrangement. The method transmits-reflects at least some of an object'semanating light through a filter component within the filter arrangementwhile the object travels through a segment of its path. The filtercomponent has a combined transmission function in which a set of simplertransmission functions is superimposed. The set includes first andsecond simpler non-uniform transmission functions and is superimposedsuch that time variation occurs in the object's emanating light inaccordance with superposition of the first and second simplernon-uniform transmission functions while the object travels through thesegment.

In specific implementations, at least one of the first and secondsimpler non-uniform transmission functions is a spectrally-dependentfunction that transmits more than one color as a function of position.The first non-uniform transmission function can be a non-periodicspectrally-dependent transmission function and the second can be aperiod transmission function. At least one of the first and secondnon-uniform transmission functions can be a spectrally-independenttransmission function that transmits black and white and/or gray scales.At least one of the simpler non-uniform transmission functions can beperiodic, random, or chirped. As above, the time variation can encodeinformation, and photosensing results can be used to obtain dataindicating the encoded information.

Implementations as described above in relation to FIGS. 1-28 could beadvantageously applied in a wide variety of sensing applications,possibly including, for example, fluorescence- or impedance-based flowcytometry or other biodetector applications that seek a signature of aparticle of unknown velocity; such biodetectors often use microfluidicchannels with inhomogeneous flow profiles, causing variation in particlevelocity. The techniques can be used to count or obtain ratios betweenfluorescing objects of different types, such as different types oftagged cells, particles, tagged DNA, and so forth. In such anapplication, calibration can be performed using known objects, e.g.,tagged beads, with known velocities to obtain template waveforms thatinclude deviations caused by fabrication tolerances but can then becompared with sensed waveforms to obtain information about unknownobjects. To improve S/N, known and sensed waveforms can be correlated,such as after time scaling of each known waveform. If a sensed waveformincludes or is accompanied by periodic modulation, a periodicity valuesuch as a frequency can be used to obtain a scaling factor for timescaling before correlation, allowing more rapid correlation than if abrute force technique is used to find a satisfactory time scaling.

Implementations described above may be advantageous in biodetectorapplications that require compact, low-cost components without criticaloptics and with high sensing efficiency. Such applications might includepoint-of-care flow cytometry (such as with an integrated flowcytometer), DNA analysis, proteomics, and so forth.

Implementations described above could successfully detect nativefluorescence differences between biological materials. Most biologicalcells are composed of only a few basic building blocks and, therefore,exhibit similar native fluorescence spectra. Interdigitated or otherwisepatchworked or patterned filter arrangements like those above areparticular suitable for differentiation of objects based on their nativefluorescence signals because the techniques are sensitive enough todetect the native fluorescence from a single cell and allow directmeasurement of distinguishing features such as intensity ratios inemission spectra. In addition, implementations of the techniques cancombine advantages of excitation and emission spectroscopy in a ruggedand compact system.

Also, implementations described above could be applied in scanning ofbio-chips or documents where objects have different emanation spectra,and so forth. The techniques may also be applicable in various low S/Nratio systems in which a known signal is bounced off an object travelingat an unknown velocity, such as where object velocity is on the order ofsignal propagation velocity, as in SONAR. The techniques may beespecially advantageous where precise information about position, speed,or type of objects is sought.

Exemplary implementations described above employ photosensors orimpedance-based sensors with specific features, but a wide variety ofsensors could be used to obtain sensing results indicating values ofvarious parameters other than emanating light intensity, parameters thatcan have time variation that indicates information about objects.Similarly, implementations described above involve sensing informationabout objects that are moving in fluidic channels or that are movingrelative to a sensor such as in scanning, but various other types offluidic implementations or other implementations in which objects movein various other ways could be sensed to obtain sensing results suitablefor techniques described above. For example, information could beobtained from native fluorescence of particles in an air stream. Also,an excitation pattern could be scanned across a glass slide withimmobilized analyte particles such as tagged cells or DNA spots, toobtain emanating fluorescent light.

Components of exemplary implementations as described above could havevarious shapes, dimensions, or other numerical or qualitativecharacteristics other than those illustrated and described above.Similarly, although the exemplary implementations generally involvesensing from a single fluidic channel, implementations could readilyinclude multiple parallel channels, allowing parallel sensing andreadout and larger scale sensing.

Some of the above exemplary implementations involve specific types offluidic components, filter components, light source components,displacement control components, sensors, and so forth, but theinvention could be implemented with a wide variety of other types ofcomponents. For example, some implementations use specific types ofspatial modulation based on one or more of an excitation pattern, afilter assembly, and/or displacement control, but various other types ofspatial modulation could be used, including any appropriate combinationof color, gray level, and black and white patterning and including otherpatterning techniques such as patterned sensing; for example, in afluidic implementation, a filter assembly or a patterned photosensorcould be printed or otherwise produced on an inward wall or otherboundary of a channel or in another appropriate location. Also, someexemplary implementations use specific types of processing, such asdigital signals obtained after converting sensed analog values. Ingeneral, however, the invention could be implemented with any suitablesignal processing techniques, including any appropriate combination ofanalog and digital processing; either or both of two compared waveformscould be obtained in analog or digital form, and any combination of timescaling could be performed before comparison. Further, some exemplaryimplementations use large area photosensors, but various ICs withphotosensing arrays might be used.

Some of the above exemplary implementations involve specific types ofemanating light, e.g. fluorescence, and specific types of excitation andfiltering suitable to fluorescent light, but these are merely exemplary.The invention could be implemented in relation to various other types ofoptical signals in various other ranges of photon energies or with anyother appropriate sensed stimuli.

Some of the above exemplary implementations involve specific materials,such as in fluidic structures with light-transmissive components or infiltering arrangements with reflective material or light blockingmaterial such as amorphous silicon, but the invention could beimplemented with a wide variety of materials and with layered structureswith various combinations of sublayers. Thicknesses of layers may varyacross any suitable range.

The exemplary implementation in FIG. 6 employs a CPU, which could be amicroprocessor or any other appropriate component. Furthermore, as notedabove, operations could be performed digitally or with analog signals,and could be done either on the same IC as a photosensor array, on othercomponents, or on a combination of the two, with any appropriatecombination of software or hardware.

The above exemplary implementations generally involve use ofencoding/sensing arrangements, sensors, photosensors, excitationarrangements, filter arrangements, displacement control arrangements,and so forth following particular operations, but different operationscould be performed, the order of the operations could be modified, andadditional operations could be added within the scope of the invention.For example, readout of sensed quantities from a sensor to obtain asensed time-varying waveform could be performed serially or in parallel,and, with an array, could be performed cell-by-cell or in a streamingoperation. Principal component analysis could be applied to specificallychosen intensity ratios in the emission spectrum in distinguishing cellsor other objects, possibly allowing identification. Multiplephotosensors along a channel could measure different intensity ratios inthe emission spectrum, possibly allowing identification of objects basedon either emission characteristics. Dyes that are very similar may bedistinguishable if they reveal only slightly different emission spectra,and use of similar dyes could be advantageous in satisfying pHrequirements within cytometers.

Discussion is now directed towards an apparatus, which is generallyreferred to as a flow cytometer, for diagnosing fluids. Examples aretesting the quality of water for personal or industrial use, or testingblood for various disease-causing organisms. According to an exemplaryembodiment, a flow cytometer produced according to the exemplaryembodiments described herein, will make use of a novel spatiallymodulated emission technique that can be used with the apparatus asshown in FIG. 29. FIG. 29 is a schematic diagram of an exemplaryembodiment of a fluidic chip for determining information about objects,particles, viruses, or functionalized micro beads (hereinafter referredto as “objects” 126), using spatial modulation and fluorescing emissionfrom moving object 126 according to an exemplary embodiment. In thespatially modulated emission technique, a patterned (e.g.,pseudo-random, periodic or chirped) mask 88 is introduced opticallybetween light emitting object 126 (light emission is stimulated by lightsource 86 not shown in FIG. 29) and a photo sensor (or photo detector)83 a to produce time variation in the signal (the signal being light,and otherwise referred to as emanating light 234). Light beam 98(generated by light source 86) impinges upon, or strikes, objects 126,and causes them to fluoresce or scatter. The act of fluorescing meansthat object 126 reacts to the impinging light beam 98, and creates lightof a different frequency and with other characteristics that are afunction of the type of object 126 that is being hit, or impinged, withlight beam 98, and its ability to fluoresce. As those of ordinary skillin the art can appreciate, not all objects 126 will fluoresce. Emanatinglight 234 is the fluorescent or scattered light leaving object 126. Asthe fluorescing or scattering objects 126 flow through excitation area87, the relative movement of object 126 produces a time modulated signalat detectors 83 a, b (the light now referred to as encoded emanatinglight 236 that leaves mask 88) due to the pattern of the provided mask88. The output of photo detector 83 a is an electrical time varyingsignal or time dependent signal as shown in FIG. 29. Correlating thedetected signal with the pattern of mask 88 discriminates thefluorescence or scatter signal (i.e., emanating light 234) from thebackground noise, giving an improved signal-to-noise discrimination, andprovides precise information about the speed and position of object 126.

According to an exemplary embodiment, a portable, hand held flowcytometer can be fabricated according to the above described principlesof spatial modulation that exhibits increased signal-to-noise ratio(SNR, or S/N) discrimination, that allows replacement of the complexoptics, fragile PMT (photomultiplier tube) detectors and bulky expensivelight sources of prior art designs. For purposes of this discussion,general reference is made to a “flow cytometer” built in accordance withthe principles discussed above. In regard to exemplary embodiments,discussion is also made in regard to prototype flow cytometer 300 andpoint-of-care flow cytometer 308, both of which are discussed in greaterdetail below. Furthermore, a flow cytometer built according to theexemplary embodiments can be both robust and miniaturized, wherein suchresult arises from the use of inexpensive components that can be readilyintegrated on a fluidic chip according to an exemplary embodiment, andby eliminating the need for sophisticated optics and critical opticalalignment. The use of lower-cost lasers, such as those used in laserpointers or recently developed laser diodes (LDs), and simpler detectionelectronics, such as miniature photodiodes and single-chip digitalsignal processors, mean that a small, rugged system can be built.

According to exemplary embodiments, techniques for using a flowcytometer fabricated as discussed herein have been demonstrated with CD4counting in whole blood. In general, a flow cytometer built according tothe exemplary embodiment comprises a disposable fluidic chip with a hoststructure, and can be used for real-time bacteria monitoring of potablewater sources to insure water quality is maintained and correctiveaction is taken quickly, as needed. As those of ordinary skill in theart can appreciate, contamination between samples can be an issue withany field testing, such as envisioned with flow cytometers 300, 308.However, through use of low cost disposable fluidic chips designed andbuilt according to the exemplary embodiments, problems associated withcontamination become substantially minimized, if not completelyeliminated.

According to a further exemplary embodiment, the flow cytometer can beused to accurately detect giardia, cryptosporidium, E.coli and bacillusendospores, among other bacteria. According to still a further exemplaryembodiment, a flow cytometer can be used to detect giardia andcryptosporidium in a handheld embodiment for use in the field, or as acompact permanently installed in-line water monitor.

According to an exemplary embodiment, the flow cytometers describedherein uses a fundamentally new design of the optical detection systemthat delivers high effective sensitivity (i.e., high signal to noise)without complex optics or bulky, expensive light sources to enable aflow cytometer that combines high performance, robustness, compactness,low cost, and ease of use.

As those of ordinary skill in the art can appreciate, commercial flowcytometers are not only expensive, but they also require sophisticatedequipment and reagents, as well as highly trained personnel to operatethem. Furthermore, in resource-limited areas, access to technicalsupport and quality assurance programs are often unavailable. Anon-exhaustive list of examples of such resource limited areas includecertain geographical localities of the world (i.e., non-industrializednations with very low per-capita income levels), rural areas inindustrialized nations with otherwise relatively high per-capita incomelevels, elderly care environments, medical units in the different armedforces when on the field, disaster relief medical units (e.g., FEMA,especially in relation to natural disaster areas, such as those ravagedby earthquakes, or hurricanes), traveling physicians (e.g., physiciansthat may be responsible for treating a relatively small amount ofpeople, but in large geographical areas), emergency medical technicians,outpatient basis clinics, groups involved in camping and exploration(e.g., polar, underwater, mountain climbing, among others), spacetravel, among other areas and types of endeavors.

One exemplary use of the flow cytometer according to an exemplaryembodiment is for rapid bacterial identification and quantitation inwater. Desired features of such a flow cytometer for use in identifyingand counting bacteria in water include detecting at least the bacterialcontaminants that include giardia, cryptosporidium, E. coli, andcacillus endospores; the flow cytometer should be a hand-held,field-deployable, fluidic-chip-based, multi-parameter flow cytometer, becapable of utilizing biochemical tagging protocol(s).

According to still a further exemplary embodiment, the flow cytometeraccording to an exemplary embodiment is a component of three tandemmicro-fluidic sub-systems: a pre-concentrator, a sample-preparationchip, and the flow cytometer. According to an exemplary embodiment, theperformance goal for the overall system is a detection sensitivity ofabout 1/100 mL. The system goal is full integration of the threesub-systems into a single compact instrument. According to still afurther exemplary embodiment, conservation of consumables is achieved byapplying the tagging protocol to only the concentrated sample volume. Aflow cytometer built according to an exemplary embodiment can beintegrated as a single hand-held point-of-care system (discussed ingreater detail below) and operated with the tagging protocol andsoftware for a complete water monitoring system, or be fabricated as anin-line monitoring system as a completely manufacturable unit (discussedin greater detail below).

According to an exemplary embodiment, the identification andquantitation of selected bacteria at the level of 1 organism per 100 mLof water sample can be accomplished at the point-of-need in less thanabout 20 minutes with the flow cytometer according to the exemplaryembodiments that can be operated by personnel with minimal training.However, the duration, identification and quantitation specificationsdiscussed above can and will vary depending upon the circumstances andnature of the environment in which the testing occurs, and as such isnot meant to be taken in a limiting manner.

According to further exemplary embodiments, the flow cytometer accordingto the exemplary embodiments can be used with at least two differentbiochemical tagging protocols: in the first instance biochemical taggingoccurs by detecting the bacteria of interest in the water sample with afluorescent moiety using antibody tags. In a second case, the tagging ofthe bacteria occurs through the use of fluorescent aptamer tags (e.g.,nucleic acid or peptide).

As those of ordinary skill in the art can appreciate, water qualitymonitoring is high priority for public, industrial, and militaryapplications. For example, microorganisms are the key catalyst forwastewater treatment, and the primary causative agents for the failureof water purification systems and the occurrence of infectious diseases.The U.S. Centers for Disease Control and Prevention (CDC) estimates thatbetween 200,000 to 1,000,000 people each year in the U.S. become illfrom contaminated drinking water, and an estimated 1,000 die. Worldwideit is estimated that 5500 people die daily from drinking contaminatedwater.

Bacterial cell quantity should be routinely monitored to maintainmicrobiological quality control of drinking water. Mobile water supplyunits currently use water purification units based on micro- orultra-filtration followed by reverse osmosis (RO) filtration and thenstores the potable water in tanks. Even with this advanced system, waterquality in the potable tanks is still a concern and needs to beregularly tested because it can become contaminated during production,handling, storage, or distribution. Due to lack of suitable testingdevices, currently all water has to be treated by an expensive andenergy consuming RO filtration technique. In order to check theintegrity of the RO water filtration system, regular micro-biologicalwater testing is desired. Other uses of a flow cytometer according tothe exemplary embodiments include water testing of swimming facilities(both public and private), including pools, and beaches, and testing ofportable and not-portable drinking water supplies. In these exemplaryuses, the flow cytometer according to the exemplary embodiments can beconfigured as a stand-alone point-of-care device, or as an in-linemonitoring device (e.g., in a pool filtration system). Currentlyavailable commercial tests can take as long as 18-24 hours to determinethe presence (or not) of micro-organisms such as E. coli. As those ofordinary skill in the art can appreciate, test results that lag that farbehind actual usage means that precious little can be done for thosethat have used or drank the affected water in the past day or so.According to further exemplary embodiments, the detection of harmfulmicroorganisms (e.g., E. coli) by a flow cytometer within about 20minutes provides a significant increase in protecting the health ofthose that drink the water, and/or swim in the water

As those of ordinary skill in the art can further appreciate, organismsthat indicate the presence of sewage and fecal contamination have beentargeted for measurement. One particular example, discussed brieflyabove, is coliform which describes a type of bacteria that includesEscherichia coli (E. coli). E. coli is generally found within theintestines of all warm blooded animals and is an indicator analyte forother dangerous pathogens. Another well known type of bacteria thatshould be detected is enterococcus, which is much like coliformbacteria, but is known to have a greater correlation withswimming-associated illnesses and is less likely to die-off in highlysaline water. These pathogens, if contacted, could result in suchsymptoms as diarrhea, cramps, and nausea.

In the US alone, billions of gallons of untreated or under-treatedsewage is discharged into waterways yearly, potentially impactingdrinking water and causing beach closings. Therefore, there is a needfor an inexpensive, early warning biosensor to monitor both beaches anddrinking water.

As those of ordinary skill in the art understand, bacterial quantitationis currently performed primarily in central laboratories with plateculture assay techniques; this infrastructure and procedures have beenintegral to microbiology for more than 100 years. The method of choiceto determine bacterial coliform count in potable water starts with themembrane filter technique, then incubation growth in a plate culturefollowed by counting of the colony-forming units. Unfortunately cultureassay techniques for quantitation are costly, labor-intensive andtime-consuming to conduct, with measurement times greater than 24 hoursdue to incubation needs. Culture-independent techniques have usedfluorescent microscopes, but results are labor intensive and subjectivebecause visual counting varies among investigators.

Presently available commercial flow cytometers are an effective andwell-established method for counting and sorting cells on a large scale,as they are rapid, sensitive, and can reliably quantify individualcells, but they are also skill and labor intensive. Commerciallyavailable flow cytometers require expensive light sources and detectorsto adequately illuminate samples and ensure that enough scattered andemitted light is collected for analysis. The complexity of the opticalsystem and the need for high-quality lasers and detectors make mostcommercial flow cytometers bulky, expensive, and fragile. Consequently,their use is limited to laboratories with highly skilled workers and afairly high level of infrastructure support. In contrast, amicro-fluidic device built according to an exemplary embodiment such asthe flow cytometer designed and built according to the exemplaryembodiments has the potential to increase ease-of-use by integratingsample pretreatment and separation strategies.

As discussed above, presently used techniques for the detection ofwaterborne parasites are primarily based on antibody-antigen reactionassays that vary in sensitivity and specificity. Also, cross-reactivitybetween pathogenic and non-pathogenic species represents a problem whenexclusively using these assays to monitor safe drinking water.

A further exemplary use of the flow cytometer according to the exemplaryembodiments is for rapid identification and quantitation of blood bornepathogens (such as bacteria or viruses), or to analyze and count theconstituents of the blood, such as certain subgroups of white bloodcells (e.g., CD4 lymphocytes). As those of ordinary skill in the art canappreciate, low levels of cluster of differentiation 4 (CD4) lymphocytesindicates a compromised immune system, and can indicate the presence ofthe human immunodeficiency virus (HIV) (which is a lentivirus, or amember of the retrovirus family), and which itself causes acquiredimmunodeficiency syndrome (AIDS). AIDS is a condition in which theimmune system of people begins to fail, leading to life-threateningopportunistic infections (i.e., pneumonia, or other illnesses).

A first exemplary embodiment of the flow cytometer according to theexemplary embodiments can be used for monitoring a single fluorescentemission band of a targeted pathogen or its signature, such as absoluteCD4 counting for HIV, by means of excitation with a laser diode (LD) orlight emitting diode (LED) and detection with a P-type intrinsic N-type(PIN) photo diode. As used herein, light source 98 includes both LD 238and LED 240, and photo detector 83 includes both PIN diode detector 122and avalanche photo diode (APD) detector 84. According to furtherexemplary embodiments, flow cytometer can also be configured as amulti-parameter instrument that can detect multiple fluorescencechannels, wherein such device can be utilized, by way of just oneexample, for percent and absolute CD4+ T-lymphocyte counts.

As with the exemplary embodiment of flow cytometer for use in watertesting discussed above, the flow cytometer according to the exemplaryembodiments for use in blood testing has the desired features ofperformance, robustness, compactness, low cost of production and use,reagent consumption, and ease of use.

A compact two-color flow cytometer according to the exemplaryembodiments can use a patterned color mask and a single large areadetector, according to an exemplary embodiment, to perform more completediagnostics, e.g., CD4% and absolute CD4 count. A further exemplaryembodiment of the flow cytometer can be used for the detection ofmalaria.

As those of ordinary skill in the art can appreciate, presentlyavailable commercial flow cytometers are indispensable tools in clinicaldiagnostics, such as in diagnosing cancer, AIDS, and infectious diseasesduring outbreaks, and also in microbiology and other areas. Chemical andor physical information is obtained about a moving object such as abiological cell, a virus, a molecule, or a sub-molecular complex, as itflows in a fluid stream. However, as those of ordinary skill in the artcan attest to, the cost, complexity, and size of existing commerciallyavailable flow cytometers preclude their use in field clinics,point-of-care (POC) diagnostics, water monitoring,agriculture/veterinary diagnostics, and rapidly deployable bio-threatdetection.

Furthermore, a number of commercially available flow cytometers usemultiple excitation sources, each focused on a well-defined location orregion separate from the others. Light emitted from each source's regionis typically analyzed with a series of beam splitters, filters, andphotomultiplier tubes (PMTs) in order to detect and distinguishdifferently stained cells or cells that concurrently carry multipledyes. Cells are typically stained in solution with different dyes priorto insertion into a cytometer, and the measurement event occurs as thecells traverse a detection region within a fluidic channel, at a speedof up to several meters per second. In the detection region, focusedlaser light (typically with an elliptical focus of 80 μm×40 μm) excitesthe dyes on the cells. The resulting fluorescent light can be collectedby a microscope lens, sorted by band pass filters, and detected by PMTsor APDs (avalanche photo diode). For each spot excitation, a respectiveset of filters and detectors is needed, which is costly and leads tobulky instruments with critical requirements to maintain opticalalignment. Since the detection region is very small, and the objectstraverse it rapidly (typical dwell times are around 10 μs), such flowcytometers have serious signal-to-noise ratio (SNR) limitations forweakly fluorescing cells. These limitations become more acute ifmultiple targets must be characterized and distinguished for counting orsorting. Thus, all presently available commercial approaches appear torequire sophisticated, high-cost components or suffer from lowperformance (e.g., time per measurement, robustness, sensitivity, easeto use).

Therefore, no commercial instrument meets all technical requirements forPOC resource-limited settings, and in particular the cost target remainsextremely challenging. In view of all this, the medical necessity andpractical challenges are enormous. The HIV pandemic has created anunprecedented global health emergency. In response, the price oflife-saving HIV drug treatment has been reduced to under $100 per year.More than 3 million people have started treatment in the past fiveyears. But of the 33 million people living with HIV worldwide, fewerthan 10% have access to CD4 cell monitoring, the critical blood testused by clinicians to decide when to start treatment. Fewer than 1% haveaccess to viral load assays, which are used for infant diagnosis and forpatient monitoring. It is estimated that about 0.6% of the world'spopulation is infected with HIV. In 2005 alone, AIDS claimed anestimated 2.4-3.3 million lives, of which more than 570,000 werechildren. The urgent need to reach HIV-infected patients presents anunprecedented opportunity to drive technology development inpoint-of-care diagnostics.

Thus, what is needed is the development of a convenient, low-costinstruments for CD4 counting that is compact and robust enough forhealthcare workers to carry to patients in remote settings. Suchportable instruments must also be capable of providing absolute CD4counts. Better information to decide when and which treatment toinitiate can be provided with CD4% (percentage of white blood cells thatare CD4+ T-lymphocytes), the CD4/CD8 ratio, or viral load measurements.The latter measurements are particularly essential for infectedchildren. According to an exemplary embodiment, flow cytometersdescribed herein have been designed to meet the medical communities'needs in fighting the global AIDs problem as discussed above.

In this section, an optical detection technique is described thatdelivers high signal-to-noise discrimination without precision opticsthat enables the flow cytometers described herein to provide highperformance, robustness, compactness, low cost, and ease of use.According to an exemplary embodiment, the enabling technique is termed“spatially modulated emission” and generates a time-dependent signal asa substantially continuously fluorescing bio-particle moves past apredefined pattern for optical transmission. Correlating the detectedsignal with the known pattern achieves high discrimination of theparticle signal from background noise. Attention is directed to FIGS.30-32

In contrast to conventional flow cytometry, wherein the size of theexcitation is restricted to approximately that of the size of theparticle, the spatial modulation technique described herein uses asubstantially larger excitation area 87 to increase the total flux offluorescence light that originates from a particle. Despite the size ofthe excitation area, the mask pattern enables a high spatial resolutionin the micron range. This allows for independently detecting andcharacterizing particles with a separation (in flow direction) that canapproach the dimension of individual particles. In addition, the conceptis intrinsically tolerant to background fluorescence originating fromfluorescing components in solution or fluorescence from the chamber orcontaminants on surfaces.

The basic technique and first proof of concept demonstration isdescribed in Ref 11 (P. Kiesel, M. Bassler, M. Beck, N. M. Johnson,Spatially modulated fluorescence emission from moving particles, Appl.Phys. Lett., 94, 041107 (2009)).

A variety of predefined masks can be used, which includes periodic,chirp, and finite-random patterns. A finite random pattern is definedherein to mean that a practical limitation has been placed, or ischaracteristic of a mask pattern; a truly random pattern would besignificantly long, if not endless. As such, any shortened mask patternbecomes less than completely random, and as a natural result, is“finite-random.” The functional form of the mask influences theobtainable particle information as well as S/N discrimination. Aperiodic mask has the advantage that the particle speed can be readilydetermined (e.g., Fourier transform or electronic lock-in techniques),however, it is less satisfactory for accurately determining absoluteposition of the particle or handling multiple particles in the detectionarea. These issues are elegantly resolved by adopting masks with apseudo-randomly defined pattern. Correlating the recorded time-varyingsignal with the mask pattern can detect multiple particles in thedetection zone and precisely determine their absolute positions andseparation, with spatial resolution related to the minimum feature sizeof the mask pattern. The combined advantages of periodic andpseudo-random masks can be obtained by integrating the two patterns in asingle mask according to an exemplary embodiment. In this case, dataanalysis can accurately yield both speed and position of each particlein real time.

In conventional flow cytometry, the size of the excitation area isrestricted approximately to the size of the particle. According to anexemplary embodiment, the method described herein uses a much largerexcitation area to increase the total flux of fluorescence light thatoriginates from a particle. Despite the large excitation area, the maskpatterning enables a high spatial resolution in the micron range. Thisallows for independently detecting and characterizing particles with aseparation (in flow direction) that can approach the dimension ofindividual particles. In addition, the concept is intrinsically tolerantto background fluorescence originating from fluorescent components insolution, fluorescing components of the chamber and contaminants on thesurface.

In FIGS. 30-32, the basic concept and benefit of imposing spatialmodulation on the fluorescence emission from a moving particle isschematically shown. The arrangement for a conventional flow cytometeris shown in FIG. 30 with fluorescence emission 74 optically excitedwithin a highly focused spot. The advantages of this approach includestrong signal, high S/N, and good particle separation. But realizingthese benefits requires high photon flux densities (i.e., intense lightsources, precision optics and critical optical alignment), with the riskof saturation effects, and accurate control of both the flow path andspeed of the particle. A conceivable partial fix for these disadvantagesis shown in FIG. 31, with large fluorescence emission 76 that increasesintegration time for emission collection. While allowing lowerexcitation flux densities, with less saturation, and eliminatingcritical optical alignment, the fluorescence signal and the S/N would beconcomitantly lower and particle separation would be poorer than in theconventional approach. The spatial modulation technique according to anexemplary embodiment is illustrated in FIG. 32 with patterned collectionzone 78 superimposed on excitation area 87. The resultant time-dependentsignal is analyzed with standard correlation techniques. This yieldsimproved S/N discrimination and high spatial resolution with neitherprecision optics nor critical alignment, while using low excitation fluxdensities. In addition, the technique yields particle speed to enablevolumetric calibration and simple fluidic handling.

Attention is now directed towards FIGS. 33, and 34, which illustrates afirst implementation of a flow cytometer (prototype flow cytometer) 300that illustrates the principles of the exemplary methods. FIG. 33illustrates, among other things, a side view of first fluidic chip 302and FIG. 34 illustrates a top view of the same first fluidic chip 302,and mask 88. The flow cytometers built in accordance with the exemplaryembodiments described herein, generally include a host structure, and afluidic chip. The host structure generally includes one or more lightsources, one or more photo detectors, and circuitry of various differentembodiments to receive, analyze and interpret the detected signals, andprovide some type of readout/display to indicate results. The hoststructure can further include various optical elements, includinglenses, filters, masks, among other items. The fluidic chip, designed tobe disposable, includes the channel for accepting analyte, that containsobjects 126 as discussed above, and further includes one or more masks,and in some cases sheath fluids and inlets/outlets for the sheath fluidsand analyte. In some cases, discussed in greater detail below, one ormore masks can be part of the host structure, or the fluidic chip canalso contain the photo detectors, and in some cases, the fluidic chipcan contain the light sources. The host circuitry can include computers,microprocessors, field programmable gate arrays, and other logiccircuitry, or combinations thereof.

Prototype flow cytometer 300, illustrated in schematic format in FIG.33, includes first fluidic chip 302, and prototype host structure 304.Prototype host structure 304, not shown in its entirety in FIG. 33,includes an optical excitation source (light source) 86, lenses 82 a, b,filter 80, and an avalanche photo detector (APD) 84 (according to anexemplary embodiment, APD 84 and PIN diode detector 122 are specificexamples of types of photo detectors 83; accordingly, in some casesreference is made to one or the other of the specific examples of photodetectors 83, but aside from certain design considerations and alsopossible for supply/demand reasons, either can be used and in generalreference may be made to either as shown in the figures. In othersituations, reference will be made to the “generic” photo detector 83).For prototype flow cytometer 300, first fluidic chip 302 was formed withtwo closely spaced quartz slides to define a flow channel about 200 μmwide and about 25 μm deep. According to an exemplary embodiment, mask88, shown in detail in

FIG. 34, includes a pattern of gratings that are about 0.1 mm in height,about 1.0 mm in length, and that can have a width as low as 20 μm. Afinite-random mask pattern (mask) 88 was photo lithographically definedin a metal film deposited on the inside surface of the top slide. Asyringe pump (not shown) can be used to control both the flow of thebead-containing solution (analyte) 96 and the sheath flow (sheath fluid)94. As those of skill in the art can appreciate, however, sheath fluid94 is not required, but can be used to optimize the light-analyteinteraction. Optical excitation can be provided by anti-resonantlycoupling laser light into first fluidic chip 302, to achieve nearlyuniform excitation along the path of the analyte 96 flow. In thisconfiguration, analyte 96 flow can be manipulated relative to theexcitation path to substantially minimize background noise and allowhigh distributed excitation with reduced bleaching of the dyes. Forexample, the interaction between the guided excitation light 98 andanalyte 96 can be restricted to the detection zone by directing the flowof analyte 96 into guided light beam 98 just before detection area 87,and directing the flow out of light beam 98 right after detection area87 (which occurs in the volume of area defined by mask 88 and APD 84).

The spatial modulation technique is described in detail in an articlepublished in the Applied Physics Letters (APL), “P. Kiesel, M. Bassler,M. Beck, N. M. Johnson, Spatially modulated fluorescence emission frommoving particles, Appl. Phys. Lett., 94, 041107 (2009)”, the entirecontents of which are incorporated herein by reference. The articlediscusses in great detail the spatial modulation technique, includingthat from the “raw” recorded data signal a correlation signal isgenerated between an ideal and the recorded data signal. The correlationsignal then indicates detection of object 126, and followingcorrelation, the derivative of the correlation signal is generated, andthis yields the position of object 126.

In order to verify the capabilities of the spatially modulated detectiontechnique and prototype flow cytometer 300, human blood samples weretested to determine the concentration of CD4 T-lymphocytes marked withR-phycoerythrin (PE) in a PBS buffer-solution. Attention is now directedtowards FIGS. 35-40.

For the blood sample testing, the set-up of prototype flow cytometer 300as shown in FIG. 33 was modified slightly. The set up used for the CD4measurements is schematically shown in FIG. 35A. Excitation light (532nm laser) 98 was focused with cylindrical lenses (not shown in FIG. 35A)at ˜20 degrees from the plane (˜70 degrees from the normal) of firstfluidic chip 302. The excitation area was about 0.6×0.03 mm². A remotesensing method was used to image the detection area of the fluidic chiponto the spatial mask (0.5×0.05 mm²) with a pseudo-random pattern and aminimum feature size of 10 μm (see FIG. 35B). Masks 88 a, b were placedin front of respective 3×3 mm² array-type avalanche photo-diodedetectors 84 a, b. The fluorescence from the CD4 cells was collected bya 20× microscope objective (NA=0.4; lenses 82 a-d). Determination ofabsolute CD4 counts, discussed in detail below, require the use of onlyone fluorescence channel (i.e., a “one” parameter instrument), and onlythe upper detector 84 a was used. For reliable detection of CD4%,however, both fluorescence channels need to be detected. For thesemeasurements, both channels were recorded simultaneously from theopposite sides of the excitation area on the fluidic chip as indicatedin FIG. 35A. This remote sensing arrangement was chosen to gain maximumflexibility for the absolute and percentage CD4 measurements (directsensing means that mask 88 is integrated into the fluidic chip and thephoto detector is attached to it; remote sensing means that particles inchannel 124 are imaged onto mask 88 with a photo detector placed orlocated behind mask 88). According to a further exemplary embodiment,CD4 measurements were successfully accomplished with the set updescribed and shown in FIGS. 33 and 34 using a spatial mask integratedinto first fluidic chip 302 using anti-resonant waveguide excitation.For these measurements, first fluidic chip 302 was redesigned to enablethe analyte stream to be directed into the guided excitation lightshortly before entering detection area 87. This minimized lightscattering from red blood cells outside detection area 87 which ishelpful when measuring whole blood samples. The excitation power densityused for the CD4 measurements was less than 100 W/cm². Theanalyte-to-sheath-flow ratio was adjusted to about 1:15, with an analyteflow rate of about 6 μl/min. For the cross section of the fluidicchannel 124 (25 μm×200 μm), an average flow speed of about 333 mm/s anda maximum flow speed v_(max) of about 543 mm/s was calculated (whichcorresponds to a pressure drop of ˜200 mbar along the 3 mm narrowsection of the channel) Measurements taken yielded v_(max) to be betweenabout 550 to about 700 mm/s for channels with a width ranging from about200 to about 250 μm, in good agreement with the prior calculations.

A basic, commercially available lab protocol was used to tag the CD4cells. The blood samples were incubated with the antibody and dye (PE)(BD reagent 555347) for about 40 minutes at room temperature and dilutedwith PBS buffer solution for dilution factors of about 1:3. No lysing ofthe red blood cells or washing steps to separate the tagged blood cellsfrom unbound dye was used.

A variety of blood samples with different dilutions (1:10 to 1:1),blood/reagent ratio, incubation times (10-40 min), and temperatures (RT,37 C) were tested. FIG. 36 shows a histogram of detected tagged cells asa function of fluorescent intensity for sample A1 with a dilution ofabout 1:5 (25 μl blood, 2 μl CD4-PE, 123 μl PBS). The plot exhibits twopeaks which are attributed to CD4 lymphocytes (the right peak) and CD4monocytes (the left peak). This result is representative of a wide rangeof measurement conditions for this donor blood (i.e., for repeatedmeasurements on the same sample and samples with modified samplepreparation). The average absolute CD4 count was about 1800 CD4 cellsper μl blood, with a variation of about ±6%. As those of ordinary skillin the art can appreciate, most deviations are probably result frommaintenance and handling of the simply-constructed first fluidic chip302 between the measurements and from sample segregation. The measuredCD4 values for this sample are at the upper end, but within the expectedrange, for human blood. The relative count rate of lymphocytes andmonocytes and, more importantly, the peak distance (intensity ratio) arein good agreement with data reported in the literature.

FIG. 37 shows the measured fluorescent amplitude for each detected cellin sample A1 as a function of particle speed for a total of about 11,000detected cells. Details on the determination of amplitude and speed aregiven above. The detected cells separate into two groups that can beassociated with CD4 lymphocytes and CD4 monocytes. Compared to the CD4lymphocytes (about 7 μm), the larger CD4 monocytes (about 15 μm) show anarrower speed distribution as flow focusing constrains them to anarrower range of flow speeds in the center of the channel. The higherminimum speed for the monocytes of ˜500 mm/s compared to ˜400 mm/s forthe lymphocytes reflects the repulsive force a cell experiences in thesteep speed gradient near a channel wall. In summary, these resultsclearly demonstrate the accuracy of the spatially modulated emissiontechnique to determine the velocity distribution of the particles.

Choosing a mask 88 with an appropriate minimum feature size will enableadditional discrimination between lymphocytes and monocytes. Smallfeatures of mask 88 will be visible in the signatures (high-frequencyfeatures) for the smaller lymphocytes, but will be weak or absent in themonocyte signals. Therefore, the signature for a particular cell can becorrelated with the expected signal for monocytes vs. lymphocytes toyield a better match. The spatially modulation technique offers severalvariables (intensity, speed, correlation) to discriminate betweenlymphocytes and monocytes.

As a particular strength of the technique, and according to an exemplaryembodiment, the correlation analysis yields the speed of each particleand can de-convolute signals from two particles that are in the sensingarea at the same time. Coincident particles can be separately detectedas long as their spatial separation is larger than the minimum featuresize of the mask. Even particles with different intensities orvelocities can be distinguished. Fluorescent beads have been detectedthat are in near coincidence with one order of magnitude difference inintensity. Those of ordinary skill in the art can appreciate that thedemonstrated capability meets the needs for important medicalapplications such as CD4 counting. FIGS. 38-40 shows a signature fromtwo closely-spaced CD4 cells. FIG. 38 is the raw data for the twoparticles; FIG. 39 illustrates both the correlation result thatindicates a first particles' position (solid line), and the expectedresult (dotted line); similarly, FIG. 40 illustrates the correlationresult that indicates a second particles' position (solid line), and theexpected result (dotted line). With conventional data processingprocedures, this event would be recorded as arising from a single cell.The correlation technique, however, clearly identifies two particles andyields their speed.

Absolute CD4+ counts can be used in the initiation and monitoring ofantiretroviral therapy (ART) in HIV-infected adults. However inpediatric patients, the percentage CD4+ T-lymphocyte to white blood cellcount value is a more useful parameter for monitoring HIV treatment,since its varies significantly less than the absolute CD4+ count. Thereis little consensus in the relevant art on which is the right method formeasuring CD4 percentage (CD4%). To facilitate demonstration ofprototype flow cytometer 300 according to an exemplary embodiment formeasuring CD4%, a standard CD4 reagent (PE-CD4, PE/CY5-CD3 and knownnumber of fluorescent micro beads), a recently introduced FACSCount CD4%reagent kit (BD model No. 339010 that consists of a single tubecontaining a mixture of three monoclonal antibodies, CD4/CD14/CD15,which were conjugated with PE/PE-Cy5/PE-Cy5, respectively), a nucleicacid dye, and a known number of fluorescent micro-beads were used. Theantibody to CD14 recognizes a human monocyte/macrophage antigen, whereasthe antibody to CD15 recognizes a human myelomonocytic antigen that ispresent on the majority of granulocytes.

Sample preparation was performed at both PARC and an externallaboratory, for comparison. The recommended sample preparation protocolwas followed. A standard two-color FACSCount reagent can be used todetermine the total number of reference beads, CD4+ lymphocytes, andCD4− lymphocytes. The new CD4% reagent identifies lymphocytes by theirDNA fluorescence and size while excluding non-lymphocytes (monocytes andgranulocytes) by their CD14+/CD15+expression. The CD4% is obtained fromindependent counts of CD4+ and CD4-lymphocytes, and the absolute CD4+ bycomparison with the known concentration of reference beads.

Attention is now directed towards FIG. 41. As those of ordinary skill inthe are can appreciate, CD4 counting can be performed with a singlefluorescence signal (e.g., from CD4-PE antibodies) on blood samples fromaverage healthy persons, there are, however, many circumstances (e.g.,co-infection with malaria, TB or other defective diseases, age ofpatient) in which distinguishing between CD4+ lymphocytes and monocytesis difficult or impossible. It is generally accepted in the medicalcommunity that for reliable CD4 diagnostics at least a two-colorcytometer is needed to reliably obtain both absolute and percentage CD4.

FIG. 41 shows representative density plots obtained from a sample ofwhole blood that was stained with the BD CD4% reagent and evaluated inprototype flow cytometer 300 using the set up described above shown inFIG. 35. For this measurement two fluorescence signals weresimultaneously recorded from the same detection area; they were recordedfrom opposite sides of first fluidic chip 302 with two array-type APDs84 as discussed in greater detail below. A volume of about 30 μl ofanalyte containing about 3 μl of whole blood (specified protocol for theCD4% reagent) was measured within approximately 5 minutes. With improveddata evaluation the data processing can be done simultaneously. As thoseof ordinary skill in the art can appreciate, accurately analyzing bloodwithin a five minute time-frame provides the capability of reducedanalysis time and/or improved data statistics.

FIG. 41 presents a pattern for the major constituents of white bloodcells (WBC) obtained from whole blood stained with BD FACSCount reagent(BD#339010). The results in FIG. 41 were obtained using the spatialmodulation technique according to an exemplary embodiment, and agreesvery well with the expected pattern measured with a state of the artflow cytometer, e.g., with a FACSCount from BD Biosciences.

For a direct one-to-one comparison, measurements using prototype flowcytometer 300 according to an exemplary embodiment were benchmarkedagainst measurements on the same samples obtained with a BD FACSCount.Excellent agreement for both absolute CD4 and CD4% was obtained betweenthe two systems. Blood samples were prepared at an external laboratorywith standard BD CD4 and CD4% protocols and reagents.

Although CD4 counting with a single fluorescence signal (e.g., fromCD4-PE antibodies) can be informative for a segment of the population ofreasonably healthy persons, there are many circumstances (e.g., age ofpatient, co-infection with malaria, TB or other infectious diseases) inwhich distinguishing between CD4+ lymphocytes and monocytes requiresadditional information. In these cases it is commonly understood that atleast a two-color cytometer is needed for reliable CD4% diagnostics.

Accordingly, a compact two-color instrument can provide more completediagnostics by including results that indicate both absolute CD4 andpercentage CD4% (or CD4/CD8 ratio). According to an exemplaryembodiment, prototype flow cytometer 300 can include two fluorescencephoto detectors 83 on opposite sides of first fluidic chip 302 with acommon excitation zone (see FIG. 35A). According to further exemplaryembodiments, light beam 98 can be introduced in the plane of firstfluidic chip 302. This can be realized either by an anti-resonantwaveguide excitation as shown in FIGS. 33, or by spot illumination at ashallow angle (e.g., by guiding the excitation light within the chip bytotal internal refection), as shown in FIGS. 35A, and 42-46 (both ofwhich are discussed in detail below). Although providing light beam 98to the excitation area 87 is more challenging when using anti-resonantwaveguide excitation, experimental demonstrations have shown that theanti-resonant waveguide excitation works in the configurations of FIGS.34, and 35 (although first fluidic chip 302 shown in FIG. 33 utilizesonly a single photo detector 83, with the correct mask 88 (e.g.patterned color mask), it too can be used for CD4% count determination).

Single parameter detection can be defined as detecting only a singlefluorescence channel which is e.g., sufficient if only one type ofobject has to be counted (e.g. absolute CD4 which requires countingtagged CD4 cells. Attention is now directed towards FIGS. 42-46 thatillustrate second fluidic chip 314 (see FIG. 42, a side view of secondfluidic chip 314, and FIG. 45, a top perspective view of second fluidicchip 314) and prototype flow cytometer 300 according to an exemplaryembodiment. Prototype flow cytometer 300 includes, as shown in FIGS. 43,44 and 46, prototype host structure 304, and second fluidic chip 314.The compact, hand-held, prototype flow cytometer 300 as shown in FIG. 43has been assembled with off-the-shelf components. The total size isabout 5×3×2 inches. The largest component is a 50 mW, 532 nm lasermodule 86 that is directed through the polished end facet (light sourceinterface 312 a) of second fluidic chip 314 onto the approximately 2×0.1mm excitation 87 area. According to an exemplary embodiment, secondfluidic chip 314 is mounted directly on detection unit 310. Detectionunit 310 can include collimator optics, a filter 80 for filtering outexcitation light 98, and a compact PIN diode detector 122 in a TO5header with integrated collection optics and integrated trans-impedanceamplifier with a gain of about 10⁷ V/A, all of which can be housedwithin housing 342 shown in FIG. 46. As discussed above, patterned mask88 can be included in second fluidic chip 314, shown in FIG. 29.Therefore, according to an exemplary embodiment, no precise alignmentbetween second fluidic chip 314 and detector unit 310 is required.

Alternatively, and according to a further exemplary embodiment, if mask88 is not part of second fluidic chip 314, then it can be made part ofsecond flow cytometer 300, and there will be self-alignment betweenprototype flow cytometer 300 and second fluidic chip 314. According to afurther exemplary embodiment, if photo detector 83 and filter 80 are notpart of second fluidic chip 314, then they can be made part of prototypeflow cytometer 300 and it will become necessary to align second fluidicchip 314 and photo detector 83 such that encoded emanating light frommask 80 will reach photo detector 83 (through filter 80; an example ofthis configuration is shown in FIG. 42). According to an exemplaryembodiment of prototype flow cytometer 300, however, the only alignmentrequired is to direct laser light beam 98 to excitation area 87, andthat alignment is relatively uncritical, because of the relatively largeexcitation area 87. Excitation area 87 is the area within which lightbeam 98 interacts with objects 126 and emanating light 234 can bedetected by photo detector 83 after passing through mask 88 and becomingencoded emanating light 236. The whole unit can be powered by batteries(e.g., two nine volt batteries and two 1.5V C-type batteries), which issufficient for at least a few hours of continuous operation).

Second fluidic chip 314 (as well as third fluidic chip 236, shown anddescribed in regard to FIG. 57), shown in FIGS. 42, 45 and 46, will becapable of being mass produced according to known manufacturingtechniques (e.g., injection molding, hot embossing, among othertechniques), the discussion of which is omitted for the dual purposes ofclarity and conciseness. According to various exemplary embodiments,different techniques can be used fabricate the fluidic components, e.g.,second fluidic chip 314 and third fluidic chip 326. For substratematerial, quartz or glass slides can be used. The fluidic channelstructure 124 of second and third fluidic chip 314, 326 can beconstructed by (micro)-structuring suitable spacer materials such as SU8photo resist, polydimethylsiloxane (PDMS) or special thin tape. Thestructuring of the spacer can be accomplished with laser processing,conventional photo lithography or micro-molding of PDMS on glass orquartz substrates. The channel structure can be sealed with cover slide,or with thermal bonding material, or gluing. As those of ordinary skillin the art can appreciate, fabrication methods to create a suitable flowcell include micro-structuring of thin adhesive tape (e.g., commerciallyavailable from 3M), with an automated laser processing tool. The tapecan then be used as spacer material and to connect substrate and coverslide. Holes for the fluidic inlet and outlet can also be fabricatedwith the laser cutter or conventional drilling. Furthermore, masks 88can be provided on a pre-structured substrate or produced duringfabrication of second fluidic chip 314. Many suppliers are capable ofintegrating masks 88 with the required spatial resolution (10 μm) andsize into second fluidic chips 314. Shown in FIG. 42, among otherdrawing figures, are several dimensions indicating approximatedimensions of several features of the fluidic chips; as those ofordinary skill in the art can appreciate, such dimensions are not meantto be in a limiting manner, and therefore should not be taken in alimiting manner, but instead represent one exemplary embodiment, andmany possible other configurations are possible and within the scope ofthe exemplary embodiments of the fluidic chips and flow cytometers.

Additional embodiments of second (and third fluidic chip 314, 326 willbe discussed in greater detail below in relation to the discussion ofpoint-of-care (POC) flow cytometer 308, in reference to differentconfigurations of masks 88, detectors 83, and filters 80.

A first exemplary embodiment of second fluidic chip 314, shown in FIG.45, includes a first and second light source interface 312 a, b, as wellas a sheath fluid inlet 90, sheath fluid outlet 91, analyte inlet 92,and outlet 93; the same inlets and outlets apply equally as well tofirst fluidic chip 302.

Inlets 90, 92 and outlets 91, 93 of second fluidic chip 314 are designedaccording to an exemplary embodiment to fluidly interface with theneedles of fluid manifold 328, which is a component of prototype flowcytometer 300. Fluid manifold 328 provides a fluidic interface betweeninput fluids (analyte and possible sheath fluids) and second fluidicchip 314. Analyte input fluid 96 flows through analyte manifold inputtube that is connected to analyte manifold input channel that is part offluid manifold 328; analyte 96 the is output from fluid manifold 328 viaanalyte manifold output needle 350 that fits within analyte inlet 92 ofsecond fluidic chip 314. A substantially similar arrangement is presentfor sheath fluid 94 (tube, channel, needle, inlet). On the output side,a similar needle for the analyte, analyte manifold output needle 350fits with analyte outlet 93, and the output analyte 96 flows throughanalyte outlet 93, analyte output needle 350, analyte manifold outputchannel 352, and analyte manifold output tube 354 to a waste collector,not shown. A substantially similar arrangement is present for the outputof sheath fluid 94. In addition, fluid manifold 328 facilitatesretention and placement of second fluidic chip 314 on detection unit 310of prototype flow cytometer 300.

For production versions of prototype flow cytometer 300 (i.e., thirdflow cytometer 308, discussed in detail below), it is expected thatlaser module can be replaced with a low-cost laser diode 238 or LED 240.Furthermore, advantage can be taken in regard to volume discounts oncomponents, and volume discounts in terms of production. Therefore, itdoes not appear unrealistic to expect a unit manufacturing cost targetfor a hand-held POC flow cytometer 308 to cost at or about $300. Theanticipated price target is extremely favorable in comparison with thatof any commercially available or publicly announced device. Additionalenhancements can include a personal digital assistant (PDA) for datacollection/evaluation, and/or a smart-phone to store/evaluate the date,and then transmit the raw and/or evaluated data to a central datacollection unit.

Referring now to FIG. 47, results are shown for measurements conductedto verify the sensitivity and dynamic range of prototype flow cytometer300. The measurements were conducted with 3.8-μm ultra-rainbowcalibration beads (Spherotech) and yielded a detection limit ˜10³ MEPE,which is sufficient for reliable CD4 counts in whole blood and alsomeets the needs for a wide range of bio-particle-detection applications.With prototype flow cytometer 300 setup for single parameter detectionand using a simple PIN diode detector 122, results for measured absoluteand percentage CD4 (CD4/CD8 ratio) in whole blood are in good agreementwith the measurements taken with a BD FACSCount from the same samples,indicating that the simple prototype made with off-the-shelf components,but using the exemplary methodology and design closely matched that ofmuch more expensive flow cytometers.

According to a further exemplary embodiment, PIN diode detector 122,located within housing 342 (FIG. 46), can be replaced by an APD orarray-type APD detector 84 (e.g., such as those manufactured byHamamatsu Photonics, K.K., or SensL, Inc.) which only slightly increasesthe footprint of prototype flow cytometer 300, but would significantlyincrease analyte 96 through-put (currently about 5 μl/min) andsensitivity. Prototype flow cytometer 300, as shown in FIGS. 43, 44 and46 with second fluidic chip 314 as shown in FIG. 45 that includes ablack/white patterned mask 88, is a single parameter flow cytometer. Bymerely replacing black/white patterned shadow mask 88 with patternedcolor mask 242, a compact (hand-held), low-cost, two-color prototypeflow cytometer 300 can be realized, and this is discussed in greaterdetail below.

The results shown in FIG. 47 clearly establish that the compact,low-cost second flow cytometer 306 can be realized by implementing thePARC spatial modulation technique according to an exemplary embodiment.Further evidence can be provided by considering that prototype flowcytometer 300 detected bacteria in the form of fluorescently stained E.coli ER2738. The signals obtained from the tagged bacteria arecomparable to the signals detected from the stained CD4 cells.

Attention is again directed to FIGS. 43-46. Prototype flow cytometer300, as shown in FIGS. 43, 44 and 46, can be used for single parameterdetection (i.e., a single fluorescence channel). For many practicalapplications, however, at least 2-4 color detection is required. Withslight modifications, prototype flow cytometer 300 can be configured toenable the detection of two fluorescence channels. For example, use offirst fluidic chip 302 as shown in FIG. 49 can simultaneously record twofluorescence signals from the same detection area at opposite sides offirst fluidic chip 302 (also as shown in FIG. 35). This has beendiscussed above in regard to two-color detection for CD4% measurements.In order to further improve the sensitivity of prototype flow cytometer300, PIN diode detector 122 and integrated amplifier, located withinhousing 342 (FIG. 46), can be replaced with an array-type APD detector84. According to an exemplary embodiment, replacement of PIN diodedetector 122 with array-type APD detector 84 can increase thesensitivity of second flow cytometer 306 by a factor of 5× to 10×, whileonly slightly increasing the footprint of the instrument.

Referring now to FIGS. 43, and 44, manifold 328 can be used foranchoring second fluidic chip 314 (as shown in FIG. 45) with up to fourfluidic ports. The different ports on second fluidic chip 314 caninclude sheath fluid inlet 90, sheath fluid outlet 91, analyte inlet 92,and fluid outlet 93 (through which both sheath fluid 94 (if used) andanalyte 96 can exit second fluidic chip 314). Second fluidic chip 314 asshown in FIG. 45 can be inserted into second flow cytometer 306, asshown in FIGS. 43 and 44, and held in place by manifold 328 and one ormore retaining screws. In addition, according to further exemplaryembodiments, syringe pumps can be connected to second fluidic chip 314of FIG. 29 to independently control the various fluidic ports (90, 91,92, 93) to realize a broad variety of fluidic schemes (i.e., use ofsheath fluid 94). Removal and replacement of second fluidic chip 314 iseasily accomplished in regard to prototype flow cytometer 300. Prototypeflow cytometer 300, as shown and described in reference to FIGS. 43, 44and 46, is a hand-held unit that can be mounted to any suitable surface.As a hand-held unit, it was substantially easy to transport it for fieldtesting.

Furthermore, according to additional exemplary embodiments, additionallaser excitation ports are available, as shown in the schematic diagramof FIG. 48. This experimental arrangement provides flexibility to applyvarious detection and excitation schemes to excitation area 87. Thegeneral schematic diagram of FIG. 48 includes second fluidic chip 314using both first and second light source interfaces 312 a, b, first andsecond light source 86 a, b, host circuitry 318 (described in detailbelow for processing the outputs of first and second photo detectors 83a, b), and host structure 304, that, in the case of prototype flowcytometer 300, as shown in FIGS. 43, 44 and 46, includes hardwaresuitable for mounting light sources 86, photo detectors 83, detectionunit 310, among other items.

Several different exemplary embodiments have been developed for twoparameter detection. A first exemplary embodiment is direct sensing. Inthe direct sensing approach, according to an exemplary embodiment, photodetectors 83 are directly mounted to the wall of channels 124 of thefluidic chip to optimize the detection scheme, first with array-type APDdetectors 84, and then with less-sensitive PIN diode detectors 122. Fortwo-color excitation (e.g., second fluidic chip 314 as seen in FIG. 42,among others), optical filters 80 are selected to block first light beam98 a from detection by, or interfering with, second photo detector 83 b(and visa-versa in regard to second light beam 98 b, and first photodetector 83 a).

A second exemplary embodiment is remote sensing. In the remote sensingapproach, objects 126 in channel 124 are imaged onto a remotelypositioned mask 88 a attached to second photo detector 83 a also, asshown in the prototype flow cytometer 300 schematic diagram of FIGS. 50,and 57. According to an exemplary embodiment the arrangement of filters80, photo detectors 83, and masks 88 can be used in both prototype flowcytometer 300 (which is primarily a demonstration vehicle), and POC flowcytometer 308 (which can be a mass-produced, retail production device).According to an exemplary embodiment, the arrangement shown in FIG. 50has the advantage of allowing patterned mask 88 a to be exchanged easilybecause they are not integrated into second fluidic chip 314 (or thirdfluidic chip 326, discussed in greater detail below). According tofurther exemplary embodiments, the remote sensing arrangement alsoallows the use of and easy-exchange of, emission (interference or color)filter 80 b, if required, to suppress light beam 98 a from reaching orbeing detected by second photo detector 83 b (and an interference filter80 a to suppress light beam 98 b from reading or being detected by firstphoto detector 83 a).

In use, prototype flow cytometer 300 can provide two or four colordetection depending on the type of masks 88 a, b that are used in secondfluidic chip 314 and within host structure 304. Light beam 98 a,generated by either laser diode 238 a (or a light emitting diode 240)can be internally reflected off first mirror 320 a after entering secondfluidic chip 314 through first light source interface 312 a. Light beam98 b enters second fluidic chip 314 b through second light sourceinterface 312 b, and is internally reflected by second mirror 320 b ;both first and second light beams 98 a,b internally reflect againstinner boundary surfaces of second fluidic chip 314 b as shown FIG. 50until they encounter objects 126 within channel 124. First and secondemanating light 234 a,b are generated, emanating in substantially alldirections. In FIG. 50, first emanating light 234 a is shown as onlyemanating upwards toward first mask 88 a and first photo detector 83 a.However, as those of ordinary skill in the art can appreciate, suchwould generally not be the case, as discussed above. Instead, firstfilter 88 a will filter substantially all, or at least a significantportion, of any emanating light 234 b that was generated from secondlight beam 98 b. Thus, for the purpose of illustration only, firstemanating light 234 a is shown as emanating towards first photo detector83 a, and second emanating light 234 b is shown as emanating towardssecond photo detector 83 b.

First emanating light passes through second fluid chip 314 and into host304 that includes lens 82. First emanating light 234 a first encounterslens 82 that collimates emanating light 234 a towards first detector 83a. First emanating light 234 then encounters first mask 88 a.

First encoded light 236 emanates from first mask 88 a, and enters firstfilter 80 a, which, as discussed above, filters all or substantially alllight generated by second light beam 98 b. Because first photo detector83 a is located remotely from fluidic channel 124, this is the “remotesensing” aspect of second fluid chip 314 and prototype flow cytometer300. The dashed lines in FIG. 50 encapsulates those components that canbe included in prototype flow cytometer 300 (and also POC flow cytometer308, as discussed in detail below). As discussed above, because firstmask 88 a is located remotely from excitation area 87 within channel124, alignment between second fluidic chip 314 and host 304 is morecritical than if only second photo detector 23 b and second mask 88 b(located on a wall of channel 124) were being used.

Second emanating light 234 b encounters second mask 88 b, producingsecond encoded light 236 b, which is filtered by second filter 80 b, andfiltered second encoded light 236 b is then detected by photo detector83 b. Second filter 80 b and second photo detector 83 b are included aspart of host 304.

Although more precise alignment between first mask 88 a and excitationarea 87 is required than with second mask 88 b and excitation area 87 insecond fluidic chip 314 as shown in FIG. 42, substantially improvedseparation is provided between the two photo detectors, meaning there isless interference (i.e., “cross-talk”) and the SNR can be improved usingthis configuration.

Attention is now directed towards FIG. 45 that illustrates secondfluidic chip 314 and FIG. 51 that illustrates POC flow cytometer 308according to an exemplary embodiment. POC flow cytometer 308 includes,as shown in FIG. 51, third host structure 324, second fluidic chip 314,and fluidic chip (chip) holder 316.

POC flow cytometer 308 is designed to be a commercial embodiment ofprototype cytometer 300 designed and built in accordance with theexemplary embodiments described herein can be used in a variety ofsettings wherein large, commercial flow cytometers are plainlyimpractical, and/or unnecessary. Several examples of such uses werediscussed in greater detail above, and need not be repeated here again.Attention is now directed towards FIG. 51, which shows POC flowcytometer 308 according to an exemplary embodiment; FIG. 45 which showsa first exemplary embodiment of second fluidic chip 314 that can belocated in chip holder 316, and FIGS. 52 and 53 that are top and sideviews of the POC flow cytometer 308 and production fluidic chip 314according to further exemplary embodiments, FIG. 54, which is a blockdiagram of second fluidic chip 314 and third flow cytometer 308 beingloaded with analyte 96, FIGS. 42, 45, 50, 55, and 56, which areschematic representations of second fluidic chip 314 according todifferent exemplary embodiments, and FIG. 57, which is a schematicrepresentation of third fluidic chip 326, are discussed in greaterdetail below.

As discussed in greater detail above, there is a great need forportable, reliable, user-friendly and inexpensive CD4 monitors forhealthcare workers in the field, as well as more sophisticated,inexpensive bench-top devices for rural clinics. The extension ofspatial modulation techniques to patterned multicolor filterarrangements according to various exemplary embodiments discussed hereinwill allow compact “high-end” point-of-care (POC) flow cytometers 308 toaddress a large variety of demanding medical applications that typicallyrequire high-performance laboratory flow cytometers capable ofmulti-parameter measurement. In conjunction with more elaborate mask 88designs (e.g., superimposed masks), substantially precisecharacterization of spectral features for (tag-free) objectidentification can be accomplished.

FIG. 51 shows a first exemplary embodiment of POC flow cytometer 308. Inuse, after taking a small analyte 96 volume (e.g., a sample of blood,not shown), a disposable production second fluidic chip 314 (that isheld by fluidic chip holder (chip holder) 316), can be inserted into POCflow cytometer 308 in such a way that excitation area 87 is locatedbetween light source 86 and photo detector 83. FIGS. 52 and 53 are topand side cut-away view of POC flow cytometer 308 and second fluidic chip314 that illustrate the interface between second fluidic chip 314, andPOC flow cytometer 308 according to an exemplary embodiment. Positioningaccuracy between POC flow cytometer 308 and second fluidic chip isrelatively uncritical, so that placement between POC flow cytometer 308and second fluidic chip 314 (and third fluidic chip 326) within about100 μm is all that is necessary for accurate counting and detection.

Substantially all of the various embodiments of second and third fluidicchips 314, 326 (third fluidic chip 326 is shown in FIG. 57) should alignchannel 124 with mask 88 to within about 10 μm; as those of ordinaryskill in the art can appreciate, such alignment tolerances can bereadily accomplished during fabrication of both second fluidic chip 314and third fluidic chip 326. FIGS. 58-60 illustrate the 10 μm alignmenttolerances: as shown in FIG. 58, a top view of channel 124 and mask 88,the 10 μm tolerance means that mask 88 should be aligned with animaginary centerline of channel 124 such that the absolute differentbetween x₁ and x₂ is less than or about 10 μm (meaning it must beparallel to the path of the centerline to within about 10 μm); as shownin FIG. 59, a side view of channel 58, mask 88 is to be positioned suchthat the absolute difference between y₁ and y₂ is less than or about 10μm (i.e., mask 88 should be parallel with respect to the imaginarycenterline to within about 10 μm); and as shown in FIG. 60, which is afront view of channel 124, in the direction of the flow of object 126,mask 88 is to be positioned such that the absolute difference between z₁and z₂ is less than or about 10 μm (i.e., mask 88 is not rotated, orplaced at an angle as shown in FIG. 60). Furthermore, according to analternate exemplary embodiment, self-alignment techniques can also beapplied to further reduce manufacturing costs if desired.

Referring again to FIG. 52, a partial side view of POC flow cytometer308 that includes third host structure 324 and second fluidic chip 314in chip holder 316 is shown. According to an exemplary embodiment, chipholder 316 is generally “T” shaped, and relatively thin. Chip holder 316can be slid into the properly sized and shaped receptacle in third hoststructure 324, and there can be a detent mechanism, or some othersimilarly operating type of mechanism, that holds/retains chip holder316 within third host structure 324. As discussed above, self-alignmentoccurs between second fluidic chip 314 and third host structure 324,such that an substantially lossless optical path if formed between lightsource(s) 86 of third host structure 324, and light interface(s) 312 ofsecond fluidic chip 314. In FIGS. 52 and 53, there are shown a first andsecond light sources, a first and second mask, and first and secondphoto detectors; this is done merely for the purpose of description, assecond fluidic chip 314 can comprise a single mask, and photo detectorand the remotely located mask 88 b does not necessarily need to beutilized. In FIG. 52, first light beam 98 a enters light sourceinterface mate 356 a that provides a substantially lossless interfacebetween the waveguide that carries first light beam 98 a and first lightsource interface 312 a of second fluidic chip 314. Once first light beam98 a enters second fluidic chop 314, it can be internally reflected viafirst mirror 320 a, or via internal reflections, as discussed in detailabove, to channel 124 and excitation area 87. First mask 88 a is shownas attached within channel 124. A substantially similar situation occurswith second light beam 98 b, which is generate by second light source 86b. FIG. 52 illustrates that there are two such light sources 86 a, b,and two such light interfaces 312 a, b, and of course both need to beoptically interfaced between the light sources and light interfaces.Both first and second light sources 86 a, b are controlled by hostcircuitry 318.

In FIG. 53, a partial cut-away side view is shown, along lines 53-53 ofFIG. 52. In this particular exemplary embodiment of second fluidic chip314, first photo detector 83 a, and first filter 80 a are located withinPOC host 324, and are aligned with excitation area 87 and first mask 88a to receive encoded emanating light 236 a from the fluorescing object126. On the bottom portion of second fluidic chip 314 shown in FIG. 53,lens 82 captures and guides emanating light 234 b from fluorescingparticle 126, and focuses it onto second filter 80 b and then throughsecond mask 88 b, thereby providing second encoded light 236 b to secondphoto detector 83 b. Filter 82, second filter 80 b, and second mask 88b, are aligned with excitation area 87 to receive emanating light 234 bfrom the fluorescing object 126. Because there are two light sources 86a, b filters 80 a, b are necessary to block out unintended light fromentering their respective photo detectors 83.

As mentioned above, the particular configurations of second fluidic chip314 can vary depending upon the intended objects to be measured; one orboth of masks 88 can be located on the fluidic chips, or one can beremotely located, as shown in FIGS. 50 and 57, or both can be remotelylocated. POC flow cytometers 308 can be fabricated to accept one or moreof the different variations of fluidic chips according to exemplaryembodiments. If one or more photo detectors are located on the fluidicchip, then an interface must be used to transfer the electrical signalsoutput from the one or more photo detectors to the host circuitry, as isdiscussed in greater detail below.

As described above, filtering of excitation light 98 can be performedwith a conventional absorption filter 80. Absorption filters aregenerally compatible with the detection mode discussed herein due to thehigh background signal tolerance of the detection method according toexemplary embodiments. Note that the measurement shown in FIG. 47 wastaken with the handheld unit (FIGS. 43 and 44) by using a conventionalcolor filter (Schott OG570). This is particular true if dyes withpronounced stokes shift are used, for instance, 530 nm excitation incombination with PE-Cy5 or PE-Cy7. Higher quality filters can also beused, or inexpensive relay optics can be introduced between detector 83and channel 124. The use of relay optics allows use ofinterference-based filters in the parallel section of the optical pathto block excitation light 98 more efficiently. The complexity and thecosts (filter, optics) added to POC flow cytometer 308 will be modest asthe relay optics can be of low quality and could even be incorporatedinto second fluidic chip 314 (or third fluidic chip 326, as shown inFIG. 57) via injection molding. According to an exemplary embodiment,and for purposes of illustration, and not limitation, BrigthLine®interference band pass filters from Semrock provide a suppression of theexcitation light of 5-6 orders of magnitude.

According to a further exemplary embodiment, there is a broad range ofapproaches to implement data acquisition, evaluation, storage, anddisplay. For example, and for purposes of illustration, and notlimitation, real-time data evaluation on FPGAs or similar chips canprovide real-time results. Alternatively, the data could beacquisitioned first into an intermediate storage and then the evaluationcould be performed downstream either with an integrated low-costprocessor or externally on a separate device such as, in furtherexemplary embodiments, a laptop, PDA or i-phone. According to stillfurther exemplary embodiments, wireless technologies can be implementedto allow for remote data acquisition and evaluation.

According to a further exemplary embodiment, there is a need to checkfor the integrity of reagents, for example, due to heat exposure,especially in resource poor environments. A first exemplary embodimentincludes the option to add suitable calibration beads to the stainingsolution that are designed to degrade at the same rate as the actualreagents. This provides a means to measure the quality of the samplepreparation, as the calibration beads would establish an internalreference that would allow POC flow cytometer 308 to qualify theaccuracy of the measurement. According to an alternative embodiment,reagents can be used that do not degrade when exposed to heat, e.g.,reagent dried out in disposable chip.

According to further exemplary embodiments, barriers can be used toprevent certain types of objects 126, in particular bio-objects, fromcoming too close to the surfaces of channel 124 in POC flow cytometer308 wherein otherwise van der Waal's forces can become problematic, andcause adhesion. Adhesion can occur when the molecules are sufficientlyclose (between about 1 nm to about 10 nm) to any surface. According tofurther exemplary embodiments, both Fluorad and polyethylene glycol(PEG) coatings work well to reduce adhesion, and others may exist thatwork even better to substantially reduce or eliminate adhesion.

FIG. 51 illustrates a preferred implementation of an exemplaryembodiment of POC flow cytometer 308 as a hand-held device. Through useof POC flow cytometer 308 as shown in FIG. 50-52, the medicalpractitioner will have a hand-held point-of-care flow cytometer thatincludes a readout unit, as well as self contained means for excitationof objects 126, detection of encoded emanating light 236, fluidichandling and data processing. According to an exemplary embodiment,analyte sample containing objects 126 is introduced to the POC flowcytometer 308 via an inserted disposable second fluidic chip 314 (orthird fluidic chip 326), which can be designed for one-time use orfinite multiple measurements. Of course, as those of ordinary skill inthe art can appreciate, multiple re-uses of second fluidic chip 314 (orthird fluidic chip 326) can lead to contamination unless due care istaken to prevent the same from happening, or wherein contamination isnot of significant concern. Dependent on a trade-off between costtargets and ease of use, second fluidic chip 314 and third fluidic chip326 can be designed to also incorporate sample preparation. The higheffective sensitivity as well as the low analyte volume willsignificantly reduce the consumption of reagents. Accordingly, aestimate of cost per test (for absolute CD4 count) can be as low as$1/test, which includes manufacturing costs of second fluidic chip 314(or third fluidic chip 326) and the reagents.

According to several exemplary embodiments of POC flow cytometer 308,there are many advantages to be realized: these include (a) high S/Ndiscrimination and distributed excitation that allows use of low costcomponents, namely, LEDs, LDs and PIN photo-diodes, to achieve highperformance, robustness, compactness, and low cost; (b) improvements influidic handling and sample preparation; (c) determination of the speedof individual particles, as well as their identity and position, toprovide true volumetric determination in a simple fluidic system with norequirement for sheath flow, which also reduces waste; (d) a robustsystem that is substantially insensitive to background noise and thatcan simplify sample preparation and waste disposal through use of wholeblood without lysing; (e) insensitivity to essentially uniformbackground noise from optical scattering and fluorescence from majorconstituents of whole blood; and (f) insensitivity to unbound dye, anduse of low or no analyte dilution.

FIG. 45 shows production fluidic chip 314 illustrating the incorporationof a spatial mask 88. However, as discussed above, spatial (black andwhite) mask 88 can easily be replaced with a patterned color mask 88,which allows for multi-color detection with a single large-area detector83. In such a configuration, the different fluorescence channel signalsare obtained by correlating the same measured time-dependent signal withthe different expected signals for each particle type, as discussed ingreater detail above. FIGS. 45 and 46 also shows that a first path ofexcitation light can be through first light source interface 312 a, anda second light source 86 b can be incorporated to input second lightbeam 98 b through second light source interface 312 b. Incorporatingfirst and second light source interfaces 312 a,b means that twodifferent excitation wavelengths can be used to create a two-wavelengthexcitation pattern in excitation area 87, or a single light source witha single excitation wavelength can be used to create an interferencepattern. For implementations using a single light source 86 to createinterference patterns, the channel geometry can be used to tailor theexpected time-modulated signal. For example, a periodic interferencepattern in combination with a tapered channel, used to create anaccelerating particle speed in the detection zone, will produce anexpected time modulated signal that is chirped rather than periodic aswould be expected from the excitation pattern alone.

Attention is now directed more specifically to FIG. 54, which is a blockdiagram of a first embodiment of second fluidic chip 314 a that can befabricated in the shape and form of second fluidic chip 314 shown inFIG. 45, and POC flow cytometer 308 being loaded with analyte 96according to different exemplary embodiments.

FIG. 54 shows a schematic of a first embodiment of production fluidicchip 314 a for the CD4 count. Disposable second fluidic chip 314 acomprises a channel 124 with an integrated mask 88 on the inner wall ofchannel 124 that is closest to photo detector 83, sample reservoir 232for holding analyte 96, waste reservoir 230 (for retaining sample liquidwith analyte 96 after being measured), and an interface to connectsecond fluidic chip 314 a to syringe 128 for drawing analyte 96 fromsample reservoir 232 through channel 124 and into waste reservoir 230.The dimensions of second fluidic chip 314 a shown in FIG. 54 (excludingpump and read-out electronics), according to exemplary embodiments, willbe approximately 2×1 cm² and the thickness will be approximately 1 mm.The sample reservoir 232 will have a capacity to hold about 30 μl ofanalyte 96. According to exemplary embodiments, disposable secondfluidic chip 314 a can be made from various types of plastics (e.g.,TOPAS, PC, or PMMA) via injection molding or hot embossing. Bothtechniques will easily meet the required tolerances (+0.1 mm positioningaccuracy of detection zone, ±2 μm for channel dimensions).

According to further exemplary embodiments, second fluidic chip 314 adepicted in FIG. 54 illustrates measurement with pretreated samples;however, sample preparation can be accomplished on disposable secondfluidic chip 314 a according to an alternate exemplary embodiment,wherein it has been demonstrated that incubation times can besubstantially reduced (by about 60×). Accordingly, advantages includesupplying reagents from reservoirs on the disposable second fluidic chip314 a so that the operator only has to draw the blood sample into acapillary, thereby reducing errors arising from improper samplepreparation. As those of skill in the art can appreciate, however, thistechnique would require more sophisticated fluidic handling which wouldadd to the cost of the disposable second fluidic chip 314 a, but wouldmake POC flow cytometer 308 more robust against operator error.

The mechanism to insert second fluidic chip 314 a into POC flowcytometer 308 can also be designed to properly engage syringe 128 (asshown in FIG. 54) and waste reservoir 232 (if not already integratedonto second fluidic chip 314 a). Spring-loaded syringe 128 (with noconsumption of battery power) can pump analyte fluid 96 through channel124 in a few minutes, as discussed in greater detail above. A releasebutton (not shown) for syringe 128 can be connected to light source 86,photo detector 83, and a real-time data evaluation system (consisting inpart of host circuitry 318, to activate start of measurements). Thereal-time data evaluation system (host circuitry 318) can furtheroptionally consist of one or more field-programmable gate array (FPGA)chips (which also can be part of host circuitry 318) and ultimatelyprovide the user with a displayed result.

FIG. 42 is a schematic diagram of second fluidic chip 314 b that can befabricated in the shape and form as shown in FIG. 45. Light beam 98enters first light source interface 312 a, and is reflected off firstmirror 320 a into to an interior surface of a light transmissive region,and then back into excitation area 87 (of channel 124) that containsobjects 126, wherein fluorescence occurs, resulting in emanating light234. Emanating light 234 is received by mask 88, wherein spatialmodulation occurs, and time varying information about objects 126 iscontained in encoded emanating light 236. A substantially similar secondfluidic chip 314 c is shown in FIG. 50 (which also can be manufacturedin the shape and form of second fluidic chip 314 as shown in FIG. 45),but this embodiment differs in that there are two light sources, 86 a,b, with different wavelengths. Time sequential use, filtering, or aslight tilt between the optical paths of both light sources can be usedto prevent unintentional cross talk or disturbance between both lasers.A second mirror 320 b reflects second light beam 98 b to the objects126, substantially like first light beam 98 a (as discussed above inregard to FIG. 42), and creates second emanating light 234 b, that isfocused by lens 82 to second mask 88 b, resulting in second encodedemanating light 236 b. Second encoded emanating light 236 b is thenfiltered by second filter 80 b, and then received by second photodetector 83 b. As with second fluidic chip 314 a of FIG. 42, the lightsources, detectors, lens, and filters are all part of POC flow cytometer308 according to an exemplary embodiment.

FIG. 55 is a schematic diagram of a fourth embodiment of second fluidicchip 314 d that can be fabricated in the shape and form as shown in FIG.45, and that utilizes a light emitting diode (LED) 240 as excitationlight source 86. According to one exemplary embodiment, a large-area,high-power LED 240 (e.g., high brightness LEDs from Osram, Cree orLuminus, area: few mm², λ˜530 nm, 5-30 W/cm²) can be positioned withinthird host structure 324 to the outside surface of one channel wall offourth fluidic chip 314 d to introduce substantially highly uniformexcitation along channel 124 over roughly one mm². In a first example offourth fluidic chip 314 d shown in FIG. 55, an off-the-shelf LED 240 wasused, even though only about 20% of the LED 240 area was useable. Inlater configurations, custom LEDs 240 with an optimized area of about0.1×1 mm² can be incorporated. Mask 88 is deposited on an inside surfaceof a wall of channel 124 on the side opposite to LED light source 240.Photo detector 83, according to a first exemplary embodiment can be aPIN diode detector 122 that includes an amplifier, and color filter 81to block extraneous excitation light 98, both of which are attached tothe outer channel wall.

As discussed, color filter 81, used in a bandpass configuration, can beused to block light beam 98 from LED 240 in the configuration where LED240 is facing photo detector 83 (it is desired that only encodedemanating light 236 be received by PIN diode detector 122). According toan exemplary embodiment, the frequency and wavelength of light beam 98will be sufficiently different from that of emanating light 234 andencoded emanating light 236, such that color filter 81 can allowsubstantially all of encoded emanating light 236 through to photodetector 83 (PIN diode detector 122), and substantially none of lightbeam 98 will pass through channel 124 and color filter 81 un-impeded.According to an exemplary embodiment, no significant differences existbetween the use of simple color filters 81, and interference filters127. The use of color filter 81 can be omitted if the lower part of thechannel is fabricated from a suitable material (color filter). Accordingto a further exemplary embodiment, the spatially modulated fluorescenceemission detection technique is intrinsically insensitive toun-modulated background light (i.e., light beam 98 from light source 86,whatever type that it might be). The depth of channel 124 in fourthfluidic chip 314 d is about 30 μm, and the mask 88 pattern is about500×50 μm², with about a 10 μm minimum feature size. The width ofchannel 124 further depends on whether an embodiment utilizing sheathflow or a sheathless implementation is used.

Sheath flow, as those of ordinary skill in the art can appreciate,involves the flow of a first fluid (the sheath fluid 94) around a secondfluid (analyte 96) that contains objects 126 that are being measured.The use of sheath fluid 94 provides many advantages including, but notlimited to, analyte focusing, and separating the channel edges fromexcitation area 87, thus avoiding or at least substantially reducing thepossibility of an additional source for scattering and backgroundfluorescence. But, as those of skill in the art can further appreciate,sheath flow can make fluidic handling relatively more complex. Accordingto an exemplary embodiment, POC flow cytometer 308 will use fourthfluidic chip 314 d that does not involve or use sheath flow.Accordingly, the width of channel 124 in fourth fluidic chip 314 d wouldbe set to be at or about the width of spatial mask 88. Still furtheraccording to an exemplary embodiment, depending upon the typicalconcentration of cells under investigation, the channel width can bechosen to maintain an average cell distance in the detection zone thatcomplies with the minimum cell distance the spatially modulatedfluorescence emission detection technique can accommodate.

Taking by way of example only, and not in any limiting sense whatsoever,in CD4 counting, the typical upper limit for particle concentration(different types of white blood cells, reference beads) to be measuredis about 8,000 cells per μl blood. Diluting the whole blood 1:2 with theantibody/dye and buffer solution and using a channel width of 50 μmwould lead to an average of 2 cells in the detection volume at any giventime during the measurement. For a pressure drop of 145 mbar along a 2mm long rectangular (30 μm×50 μm) channel, the average flow speed wouldbe about 0.33 m/s, and the throughput of the device would be 0.5 μl/s.For a statistically relevant initial blood sample of 10 μl (dilution1:2→30 μl analyte), the total time for the measurement would be about 60seconds. According to an exemplary embodiment, these measurements wereperformed with array-type APD detectors 84, and PIN diode detectors 122in combination with a high-end, low noise amplifier (e.g.,http://www.femto.de/index.html) that can provide the requiredamplification of about 2×10⁷, with a sampling rate of about 200 kHz.According to further exemplary embodiments, however, a low costtrans-impedance amplifier can be used that would require the reductionof the particle speed to be at or about 0.11 m/s and which will increasethe time per test to about 180 seconds, or about three minutes. Furtherstill, according to another exemplary embodiment, the measurements canalso be preformed with little or no PBS dilution. According to aexemplary embodiment, minimizing the use of PBS dilution eases samplepreparation and reduces uncertainty of the mixing ratio.

According to an exemplary embodiment, one advantage of the abovedescribed approach is the simplicity of the fluidic handling. Since thespatially modulated fluorescence emission detection technique yieldsconcurrently both the fluorescent intensity of the particles and theirspeed distribution, it is possible to determine the actual flow rateand, therefore, the analyte volume from the data. Consequently,quantitative measurements do not require accurate flow control, andrather simple fluidic handling techniques can be used. This is inparticular true for sheath-less implementations. Using sheath fluid 94can introduce errors due to fluctuations of the ratio between sheathfluid 94 and analyte 96 and, therefore, requires more elaborate designof the fluidic channel 124. According to an exemplary embodiment, and asshown in FIG. 54 (discussed in greater detail above), fluidic handlingcan be as simple as connecting a spring operated syringe 128 to analyteoutlet 93 and drawing analyte 96 from open well 232 through fluidicdevice 302 and channel 124 into waste reservoir 230. For applicationswhere the duration of the measurement is less critical, according tofurther exemplary embodiments, gravity or capillary forces can be usedto move analyte 96 through channel 124. According to a further exemplaryembodiment, PIN diode detector 122 can be connected to amicro-processor, or a field-programmable gate array device (not shown)and data evaluation (through various algorithms) can be performedconcurrently with the measurement.

Through appropriate selection and design of mask 88, individual cellscan be detected with the spatially modulated fluorescence emissiondetection technique. According to an exemplary embodiment, the minimumfeature size of mask 88 is between about 10 μm to about 20 μm, and mask88 is placed in close proximity to the cells in channel 124. As shown inthe configurations of FIGS. 42 and 56 (FIG. 56 is discussed in detailbelow), mask 88 is deposited on an inside wall of channel 124. Due tothe channel depth, about 30 μm, and the flow profile, the cells traversethe mask 88 at typical distances of between about 25 μm and about 5 μm.Accordingly, mask 88 will effectively modulate the fluorescent intensityrecorded by PIN diode detector 122. It is important to note that thelateral alignment of PIN diode detector 122 and light source 86 withregard to channel 124 is not critical. The area of light source 86 andphoto detector 83 simply need to overlap with the whole detection areadefined by mask 88. According to an exemplary embodiment, this permitsthe use of disposable fourth fluidic chip 314 d (or any of the otherembodiments of second fluidic chip 314, or third fluidic chip 326) withrather low production tolerances. The required position accuracy betweenfourth fluidic chip 314 d, photo detector 83, and light source 86 isonly about 0.1 mm. Therefore, fourth fluidic chip 314 d can be insertedbetween photo detector 83 and light source 86 by using simpleself-aligning conical alignment marks.

Referring again to FIGS. 55 and 56, it can be seen that, according to anexemplary embodiment, the electro-optical components (filter 80, photodetector 83, and light source 86) are located in third host structure324, and juxtaposed with the disposable fourth fluidic chip 314 d (orfifth fluidic chip 314 e) by inserting fourth fluidic chip 314 d/fifthfluidic chip 314 e into third host structure 324 (third host structure324 can also be referred to as the “reader” as it contains theelectro-optical components and signal processing circuitry according toa preferred exemplary embodiment). Since light source 86 and photodetector 83 are part of reader/host structure 324 and are notdisposable, the cost constraints on these components are not as severeas for fourth fluidic chip 314 d/fifth fluidic chip 314 e. Accordingly,higher performance components can be used, and the inclusion ofinexpensive relay optics as warranted. According to a further exemplaryembodiment, relay optics (mirror) 320, as shown in FIG. 56, can be partof third host structure 324. Use of mirrors 320 provides for imaging ofthe high-brightness LED 240 onto excitation area 87 at an angle andstrongly reduces the required filter performance since light source 86(in this case LED 240) is no longer directly facing photo detector 83.According to a further exemplary embodiment, mirrors 320 can be madepart of fifth fluidic chip 314 e and be disposable as well.

FIG. 49 is a schematic diagram of a sixth embodiment of second fluidicchip 314 f according to an exemplary embodiment that contains a firstphoto detector 83 a, and a second photo detector 83 b. First photodetector 83 a can detect and distinguish different types of objects 126,and second photo (or additional detectors) 83 b can be placed along theparticle path to measure different absorption ratios. According to anexemplary embodiment, a first significant advantage of the techniquedescribed herein compared to conventional multi-color flow cytometry isthat in the technique described herein, different light sources 86excite analyte 96 at almost the same time and location and that isconducive to measuring differences in the excitation spectra with veryhigh precision. According to further exemplary embodiments,substantially all errors induced by time-dependent factors such asbleaching, intermixing, and diffusion, as well as errors induced bydifferences in the excitation spot such as temperature gradients, andoptical misalignment, are substantially eliminated.

Attention is directed towards FIGS. 61-63, which illustrate the basicconcept of multicolor emission, and further illustrate a seventhembodiment of second fluidic chip 314 g that can be manufactured in thesame shape and form as second fluidic chip 314 as shown in FIG. 45. Formulti-color emission detection, shadow mask 88 with binary transmissionof 1 or 0) is replaced with patterned color band pass filter masks(color mask) 242 in order to be selective to different wavelengthsranges. FIG. 61 shows an exemplary embodiment of second fluidic chip 314g that allows detection of two colors (e.g., red and green) withpseudo-random patterns of red and green band pass filters. The red andgreen color mask 242 is shown in FIG. 62. According to a furtherexemplary embodiment, and dependent on the application, it can befavorable to use a pattern containing multiple band pass filters inorder to allow for the simultaneous detection of multiple colors in theexcitation area 87. In this configuration, the modulation offluorescence optical output signal is caused by the specific spectralemission of object 126, and therefore, is necessarily color sensitive.The modulation depth (i.e., the difference between the non-normalizedhigh and low intensities of the received encoded emanating light 236),as well as the resulting pattern of the output signal, containwavelength information. For example, in second fluidic chip 314 g thatuses color mask 242 as shown in FIG. 62, red and green emitting objects126 a can produce complementary modulated patterns as shown in FIG. 63.In such a case the object color can be identified based on informationobtained by correlation and, therefore, allows for reliablediscrimination with high SNR after data processing in comparison to theSNR in the measured signal. Said another way, information contained inthe absorption or emission spectrum of object 126 is encoded in thetime-dependent signal. By using array-type detector 84 (e.g. array ofpixilated APD as offered by SensL) combined with different spatiallymodulated color masks 242, many different objects 126 can bedistinguished. Referring to FIGS. 62 and 63, note that for areas in FIG.62 that show green band-pass areas (e.g., at the very left-most portionof color mask 242, in FIG. 62 (labeled “A”)), there is a correspondinggreen object signal in FIG. 63 (e.g., at the very left most portion ofthe output signal diagram shown in FIG. 63 (labeled “A′”)). The “B[ and∓B′” designations point to a red band-pass area, and red object signal,respectfully.

According to an exemplary embodiment, multicolor excitation is realizedby changing the wavelengths of the excitation within excitation area 87.Along the path of object 126, the wavelength is changed with a defined(periodic, chirped or pseudo-random) pattern. The resultingtime-dependent signal (i.e., encoded emanating light 236), mainlyreflects the excitation (absorption) difference of object 126 withrespect to the different excitation wavelengths. For example, considertwo types of objects 126 that absorb at two different excitationwavelengths. If the objects 126 a, b have identical or similar emissionbehavior (i.e., efficiency and wavelength), this will result incomplementary time-dependent detector signals similar to that shown inFIG. 63. An exemplary embodiment would be the combination of the dyesPacific Orange and phycoerythrin (PE) that can be excited with 405 nmand 488 nm excitation lasers (i.e., light beams 98 a, b), respectively.The emission of these dyes can be detected in the yellow/orange spectralrange (e.g. 585 and 540 nm spectral ranges). Since the modulation depthof the signal depends on the absorption contrast at the two excitationwavelengths, even multiple particles exhibiting different absorptioncontrasts can be distinguished.

FIG. 57 is a schematic diagram of third fluidic chip 326 that can befabricated in the shape and form of second fluidic chip 314 shown inFIG. 45. First light beam 98 a enters first light source interface 312a, and is reflected off first mirror 320 a and is then subsequentlyreflected off an interior surface of a light transmissive region, andback into excitation area 87 that contains objects 126, whereinfluorescence occurs, resulting in first emanating light 234 a. Firstemanating light 234 a is received by first mask 88 a, wherein spatialmodulation occurs, and time varying information about objects 126 iscontained in first encoded emanating light 236 a. First encodedemanating light 236 a and any extraneous light from second light source86 b is then filtered by first filter 80 a. A second light source 86 b,with a different wavelength, transmits second light beam 98 b, and asecond mirror 320 b reflects second light beam 98 b to the objects 126,substantially similar to first light beam 98 a (as discussed above), andcreates second emanating light 234 b, that is focused by lens 82 tosecond mask 88 b, resulting in second encoded emanating light 236 b.Second encoded emanating light 236 b and any extraneous light from firstlight source 86 a is then filtered by second filter 80 b, and thenreceived by second photo detector 83 b. Both first and second lightsources 86 a, b, lens 82, and second filter are all part of POC flowcytometer 308 according to an exemplary embodiment. First detector 83 a,first filter 80 a, and first connector 322 a are components of thirdfluidic chip 326. First connector 322 a connects to second connector 322b to carry the electrical signals from first photo detector 83 a to hostcircuitry 318.

While the invention has been described in conjunction with specificexemplary implementations, it is evident to those skilled in the artthat many alternatives, modifications, and variations will be apparentin light of the foregoing description. Accordingly, the invention isintended to embrace all other such alternatives, modifications, andvariations that fall within the spirit and scope of the appended claims.

1. An article of manufacture comprising: a fluid-engaging structure,wherein the fluid-engaging structure includes (a) a channel that in usecan contain fluid and through which objects can move; (b) one or morebounding parts that bound the channel, and wherein at least one of theone or more bounding parts include one or more light transmissiveportions, wherein at least one of the one or more light transmissiveportions is configured to receive excitation light and provide thereceived excitation light, and wherein excitation light enters thechannel and interacts with an object resulting in emanating light; and(c) one or more mask arrangements configured to receive at least part ofthe emanating light and in response, provide encoded emanating light,wherein the one or more mask arrangements and the channel are furtherconfigured so that the encoded emanating light includes time variationresulting from relative movement between the one or more maskarrangements and the object, the time variation including informationabout the object.
 2. A method of using the article of manufactureaccording to claim 1, the method comprising: causing an object to movethrough the channel in a fluid; causing the excitation light to enterthe channel through the one or more light transmissive portions andinteract with the object, resulting in the emanating light from theobject; receiving the emanating light at a first of the one or more maskarrangements; and providing the encoded emanating light with the timevariation including the information about the object in response to thereceived emanating light.
 3. The method according to claim 2, furthercomprising: receiving the encoded emanating light at a photosensitivesurface of a large area photosensor; and providing an electrical signalby the large area photosensor, in response to the received encodedemanating light, the electrical signal indicating one or more sensedtime-varying waveforms, and wherein at least one of the one or moresensed time-varying waveforms indicating the information about theobject, and wherein, a host structure includes the large areaphotosensor, the host structure being separate from and useable with thefluid-engaging structure; and operating circuitry in the host structureto respond to the electrical signal by providing data indicating theinformation about the object.
 4. The method according to claim 3,wherein the host structure includes one of a handheld device, thehandheld device configured to monitor discrete samples of fluid andobjects, and an in-line device, the in-line device configured tosubstantially continuously monitor the fluid and objects.
 5. The articleof manufacture according to claim 1, wherein the fluid-engagingstructure further comprises: a first of the one or more maskarrangements configured to receive at least part of the emanating lightand in response provide encoded emanating light to received emanatinglight, and is located on a first portion of the bounding parts; one ormore mirrors, at least one of the one or more mirrors configured tosubstantially reflect the excitation light through the one or more lighttransmissive portions; and an internal reflecting surface, wherein theinternal reflecting surface is configured to re-transmit the reflectedexcitation light into the channel with the fluid and objects, andwherein the article of manufacturing is configured for use with a hoststructure, and wherein the host structure includes: at least oneexcitation light source; a filter configured to receive and filter theencoded emanating light; and a photo-sensor configured to receive thefiltered encoded emanating light and detect time variation resultingfrom relative movement between the one or more mask arrangements and theobjects, the time variation including information about the objects, andwherein the photo-sensor is further configured to provide an electricalsignal indicating one or more sensed time-varying waveforms.
 6. Thearticle of manufacture according to claim 1, wherein the article ofmanufacturing is configured for use with a host structure, and whereinthe host structure comprises: at least one excitation light source; atleast one filter configured to receive and filter the encoded emanatinglight; and a photo-sensor configured to receive the filtered encodedemanating light and detect time variation resulting from relativemovement between the one or more mask arrangements and the objects, thetime variation including information about the objects, and wherein thephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light are located on a first exterior sideof the one or more bounding parts of the fluid-engaging structure, andthe at least one excitation light source is located substantiallydirectly opposite that of the photo-sensor and the at least one filterconfigured to receive and filter the encoded emanating light.
 7. Thearticle of manufacture according to claim 1, wherein the article ofmanufacturing is configured for use with a host structure, and whereinthe host structure comprises: at least one excitation light source; atleast one filter configured to receive and filter the encoded emanatinglight; and a photo-sensor configured to receive the filtered encodedemanating light and detect time variation resulting from relativemovement between the one or more mask arrangements and the objects, thetime variation including information about the objects, and wherein thephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light are located on a first exterior sideof the one or more bounding parts of the fluid-engaging structure, andthe at least one excitation light source is located substantiallyadjacent to that of the photo-sensor and the at least one filterconfigured to receive and filter the encoded emanating light.
 8. Thearticle of manufacture according to claim 1, wherein the fluid-engagingstructure further comprises: at least two mirrors, wherein a firstmirror is configured to substantially reflect first excitation lightfrom a first excitation light source through the one or more lighttransmissive portions, and a second mirror is configured tosubstantially reflect second excitation light from a second excitationlight source through the one or more light transmissive portions; aninternal reflecting surface, wherein the internal reflecting surface isconfigured to re-transmit the first reflected excitation light into thechannel with the fluid and objects, and the internal reflecting surfaceis configured to re-transmit the second reflected excitation light intothe channel with the fluid and objects, and wherein the re-transmittedfirst and second excitation light interacts with the object resulting ina combined emanating light, wherein a first of the one or more maskarrangements is located on a first portion of the bounding parts,wherein, the first of the one or more mask arrangements is configured toreceive at least part of the combined emanating light at a first rangeof photon energies and, in response, provide encoded combined emanatinglight at the first range of photon energies; and a host structure, thehost structure including the first excitation light source configured totransmit the first excitation light; the second excitation light sourceconfigured to transmit the second excitation light; a firstphoto-sensor; a second photo-sensor; a second of the one or more maskarrangements, and wherein the second of the one or more maskarrangements is configured to receive at least part of the combinedemanating light and, in response, provide encoded combined emanatinglight at a second range of photon energies; a first filter locatedsubstantially adjacent to the first photo-sensor, wherein the firstfilter is configured to pass a first portion of the encoded combinedemanating light that corresponds to the first range of photon energiesto the first photo-sensor; and a second filter located substantiallyadjacent to the second photo-sensor, wherein the second filter isconfigured to pass a second portion of the encoded combined emanatinglight that corresponds to the second range of photon energies to thesecond photo-sensor, and wherein, the first photo-sensor is configuredto receive the filtered first portion of the encoded combined emanatinglight that corresponds to the first range of photon energies and detecttime variation resulting from relative movement between the one or moremask arrangements and the objects, the time variation includinginformation about the objects, and wherein the first photo-sensor isfurther configured to provide a first set of electrical signalsindicating one or more sensed time-varying waveforms to host structurecircuitry, and the second photo-sensor is configured to receive thefiltered second portion of the encoded combined emanating light thatcorresponds to the second range of photon energies and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a second set of electrical signals indicating oneor more sensed time-varying waveforms to host circuitry.
 9. The articleof manufacture according to claim 1, wherein the fluid-engagingstructure further comprises: a first receptacle for accepting the fluid,and the objects within the fluid; a second receptacle for accepting thefluid and the objects within the fluid following passage through thechannel; and a vacuum spring-loaded syringe configured to compelmovement of the fluid and the objects through the channel, and toenergize an excitation light source upon release of the spring.
 10. Thearticle of manufacture according to claim 1, wherein the fluid-engagingstructure comprises: a first of the one or more mask arrangements isconfigured to receive at least part of the emanating light and, inresponse, provide color-dependent encoded emanating light and is locatedon a first portion of the bounding parts, and wherein the fluid engagingstructure is configured for use with a host structure, and wherein thehost structure includes: at least one excitation light source; a filterconfigured to receive and filter the color-dependent encoded emanatinglight; and a photo-sensor configured to receive the filteredcolor-dependent encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the photo-sensor is further configured toprovide an electrical signal indicating one or more sensed time-varyingwaveforms.
 11. The article of manufacture according to claim 1, whereinthe fluid-engaging structure comprises: a first of the one or more maskarrangements configured to receive at least part of the emanating lightand, in response, provide first color-dependent encoded emanating lightand is located on a first portion of the bounding parts; a second of theone or more mask arrangements configured to receive at least part of theemanating light and, in response, provide second color-dependent encodedemanating light and is located on a second portion of the boundingparts, and wherein the fluid engaging structure is configured for usewith a host structure, and wherein the host structure includes: a firstexcitation light source; a second excitation light source; a firstfilter configured to receive and filter the first color-dependentencoded emanating light, the first filter located substantially adjacentto the first of the one or more mask arrangements; a second filterconfigured to receive and filter the second color-dependent encodedemanating light, the second filter located substantially adjacent to thesecond of the one or more mask arrangements; a first photo-sensorlocated substantially adjacent to the first filter, and wherein thefirst photo-sensor is configured to receive the first filteredcolor-dependent encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the first photo-sensor is furtherconfigured to provide a first electrical signal indicating one or moresensed time-varying waveforms; and a second photo-sensor locatedsubstantially adjacent to the second filter, and wherein the secondphoto-sensor is configured to receive the second filteredcolor-dependent encoded emanating light and detect time variationresulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a second electrical signal indicating one or moresensed time-varying waveforms.
 12. An article of manufacture comprising:a host structure, wherein the host structure includes (a) at least oneexcitation light source; (b) one or more support parts structured tosupport a fluid-engaging structure on the host structure, wherein thefluid-engaging structure includes a channel that in use can containfluid and through which objects can move; one or more bounding partsthat bound the channel, and wherein at least one of the one or morebounding parts include one or more light transmissive portions, whereinat least one of the one or more light transmissive portions isconfigured to receive excitation light and provide the receivedexcitation light, and wherein excitation light enters the channel andinteracts with an object resulting in emanating light; and one or moremask arrangements configured to receive at least part of the emanatinglight and, in response, provide encoded emanating light, and wherein theone or more mask arrangements and the channel are further configured sothat the encoded emanating light includes time variation resulting fromrelative movement between the one or more mask arrangements and theobject, the time variation including information about the object; (c)at least one filter configured to receive and filter the encodedemanating light; (d) at least one photo-sensor configured to receive thefiltered encoded emanating light and detect time variation resultingfrom relative movement between the one or more mask arrangements and theobjects, the time variation including information about the objects, andwherein the photo-sensor is further configured to provide a firstelectrical signal indicating one or more sensed time-varying waveforms;and (e) circuitry configured to receive the first electrical signals andwhich is further configured to provide second electrical signals inresponse to the first electrical signals resulting from photosensing ofthe encoded emanating light, and wherein the second electrical signalsindicate the information about the objects.
 13. The article ofmanufacture according to claim 12, wherein the photo-sensor and the atleast one filter configured to receive and filter the encoded emanatinglight are located on a first exterior side of the one or more boundingparts of the fluid-engaging structure, and the at least one excitationlight source is located substantially directly opposite that of thephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light.
 14. The article of manufactureaccording to claim 12, wherein the at least one photo-sensor and the atleast one filter configured to receive and filter the encoded emanatinglight are located on a first exterior side of the one or more boundingparts of the fluid-engaging structure, and the at least one excitationlight source is located substantially adjacent to that of thephoto-sensor and the at least one filter configured to receive andfilter the encoded emanating light.
 15. The article of manufactureaccording to claim 12, wherein the host structure further comprises: afirst excitation light source configured to transmit first excitationlight; a second excitation light source configured to transmit thesecond excitation light, and wherein the fluid-engaging structure isconfigured to provide combined emanating light resulting frominteraction between the first excitation light and the objects, and thesecond excitation light and the objects, and wherein the fluid-engagingstructure further includes a first of the one or more mask arrangementsthat is located on a first portion of the bounding parts, and wherein,the first of the one or more mask arrangements is configured to receiveat least part of the combined emanating light at a first range of photonenergies and, in response, provide encoded combined emanating light; afirst photo-sensor; a second photo-sensor; a second of the one or moremask arrangements, and wherein the second of the one or more maskarrangements is configured to receive at least part of the combinedemanating light and, in response, provide encoded combined emanatinglight at a second range of photon energies; a first filter locatedsubstantially adjacent to the first photo-sensor, wherein the firstfilter is configured to pass a first portion of the encoded combinedemanating light that corresponds to the first range of photon energiesto the first photo-sensor; and a second filter located substantiallyadjacent to the second photo-sensor, wherein the second filter isconfigured to pass a second portion of the encoded combined emanatinglight that corresponds to the second range of photon energies to thesecond photo-sensor, and wherein the first photo-sensor is configured toreceive the filtered first portion of the encoded combined emanatinglight that corresponds to the first range of photon energies and detecttime variation resulting from relative movement between the one or moremask arrangements and the objects, the time variation includinginformation about the objects, and wherein the first photo-sensor isfurther configured to provide a first set of electrical signalsindicating one or more sensed time-varying waveforms to host structurecircuitry, and the second photo-sensor is configured to receive thefiltered second portion of the encoded combined emanating light thatcorresponds to the second range of photon energies and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a second set of electrical signals indicating oneor more sensed time-varying waveforms to host circuitry.
 16. The articleof manufacture according to claim 12, wherein the filter is furtherconfigured to receive and filter color-dependent encoded emanatinglight, and wherein the photo-sensor is further configured to receive thefiltered color-dependent encoded emanating light and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the photo-sensor is further configured toprovide an electrical signal indicating one or more sensed time-varyingwaveforms.
 17. The article of manufacture according to claim 12, whereinthe host structure comprises: a first excitation light source; a secondexcitation light source; a first filter configured to receive and filterfirst color-dependent encoded emanating light provided by thefluid-engaging structure, the first filter located substantiallyadjacent to a first of the one or more mask arrangements located on thefluid-engaging structure; a second filter configured to receive andfilter second color-dependent encoded emanating light provided by thefluid engaging structure, the second filter located substantiallyadjacent to a second of the one or more mask arrangements located on thefluid-engaging structure; a first photo-sensor located substantiallyadjacent to the first filter, and wherein the first photo-sensor isconfigured to receive the first filtered color-dependent encodedemanating light and detect time variation resulting from relativemovement between the one or more mask arrangements and the objects, thetime variation including information about the objects, and wherein thefirst photo-sensor is further configured to provide a first electricalsignal indicating one or more sensed time-varying waveforms; and asecond photo-sensor located substantially adjacent to the second filter,and wherein the second photo-sensor is configured to receive the secondfiltered color-dependent encoded emanating light and detect timevariation resulting from relative movement between the one or more maskarrangements and the objects, the time variation including informationabout the objects, and wherein the second photo-sensor is furtherconfigured to provide a third electrical signal indicating one or moresensed time-varying waveforms, and wherein the circuitry is furtherconfigured to receive the first electrical signals and the thirdelectrical signals, and which is further configured to provide fourthelectrical signals in response to the first and third electrical signalsresulting from photosensing of the encoded emanating light, and whereinthe fourth electrical signals indicate the information about theobjects.
 18. The article of manufacture according to claim 12, whereinthe host structure further comprises: a slot, wherein the slot isconfigured to receive the fluid-engaging structure, to provide asubstantially lossless light-interface between a first excitation lightsource and the fluid engaging structure, and to further provide asubstantially lossless light-interface between a first photosensor andthe fluid engaging structure.
 19. The article of manufacture accordingto claim 18, wherein the host structure further comprises: a secondphotosensor; a second excitation light source, and wherein the slot isconfigured to provide a substantially lossless light-interface betweenthe second excitation light source and the fluid engaging structure, andto further provide a substantially lossless light-interface between thesecond photosensor and the fluid engaging structure; an interface boardconfigured to accept data entries by a user of the article ofmanufacture; and a display, wherein the display is configured to displaydata about the fluid to the user of the article of manufacture, andwherein the host structure is further configured to be one of either ahand-held unit, or an in-line unit.
 20. A method of using the article ofmanufacture according to claim 12, the method comprising: receiving thefluid-engaging structure, wherein the fluid engaging structure includesthe fluid and objects; determining information about the objects withinthe fluid; and displaying the information about the objects within thefluid to the user of the article of manufacture.
 21. The method of usingthe article of manufacture according to claim 20, wherein the step ofreceiving the fluid-engaging structure comprises: locating thefluid-engaging structure in a slot in the host structure, wherein theslot is configured to secure temporarily the fluid-engaging structure tothe host structure, provide a first light-interface between a firstexcitation light source on the host structure and the fluid engagingstructure, and further provide a second light-interface between a firstphotosensor on the host structure and the fluid engaging structure, andwherein substantially no fluid in the fluid-engaging structure contactsthe host structure, and wherein the step of determining informationabout the objects within the fluid comprises: receiving filtered encodedemanating light from the fluid-engaging structure by a photo-sensor,wherein the encoded emanating light includes time variation resultingfrom relative movement between the one or more mask arrangements of thefluid-engaging structure and the object, the time variation includinginformation about the object; detecting time variation resulting fromrelative movement between the one or more mask arrangements and theobjects by the photo-sensor; providing a first electrical signal fromthe photo-sensor that indicates one or more sensed time-varyingwaveforms; and receiving by host circuitry the first electrical signals;and providing second electrical signals in response to the firstelectrical signals, wherein the second electrical signals indicate theinformation about the objects, and further wherein the informationincludes at least one of a type of the object, a quantity of theobjects, a velocity of the objects, and a color of the objects.
 22. Anarticle of manufacture comprising: a host structure, wherein the hoststructure includes a first excitation light source; a second excitationlight source; one or more support parts structured to support afluid-engaging structure on the host structure, wherein thefluid-engaging structure includes a channel that in use can containfluid and through which objects can move; one or more bounding partsthat bound the channel, and wherein at least one of the one or morebounding parts include one or more light transmissive portions, whereinat least one of the one or more light transmissive portions isconfigured to receive excitation light and provide the receivedexcitation light, and wherein excitation light enters the channel andinteracts with an object resulting in emanating light; a first maskarrangement configured to receive at least part of the emanating lightand, in response, provide encoded emanating light, and wherein the firstfilter arrangement and channel are further configured so that theencoded emanating light includes time variation resulting from relativemovement between the first mask arrangement and the object, the timevariation including information about the object; a first filterconfigured to receive and filter the encoded emanating light from thefirst mask arrangement; and a first photo-sensor configured to receivethe filtered encoded emanating light from the first filter and detecttime variation resulting from relative movement between the one or moremask arrangements and the object, the time variation includinginformation about the objects, and wherein the first photo-sensor isfurther configured to provide a first electrical signal indicating oneor more sensed time-varying waveforms; a first electricalinterconnection; and a second mask arrangement configured to receive atleast part of the emanating light, and, in response, provide encodedemanating light, and wherein the second mask arrangement and channel arefurther configured so that the encoded emanating light includes timevariation resulting from relative movement between the second maskarrangement and the object, the time variation including informationabout the object; a second filter configured to receive and filter theencoded emanating light from the second filter arrangement; and a secondphoto-sensor configured to receive the filtered encoded emanating lightfrom the second filter and detect time variation resulting from relativemovement between the second mask arrangement and the object, the timevariation including information about the object, and wherein the secondphoto-sensor is further configured to provide a third electrical signalindicating one or more sensed time-varying waveforms; and circuitryconfigured to receive the first and third electrical signals and whichis further configured to provide fourth electrical signals in responseto the first and third electrical signals resulting from photosensing ofthe encoded emanating light, and wherein the fourth electrical signalsindicate the information about the object.